Fenfluramine compositions and methods of preparing the same

ABSTRACT

Methods of preparing a fenfluramine active pharmaceutical ingredient are provided. Aspects of the method include (a) hydrolyzing a 2-(3-(trifluoromethyl)phenyl)acetonitrile composition to produce a 2-(3-(trifluoromethyl)phenyl)acetic acid composition; (b) reacting the 2-(3-(trifluoromethyl)phenyl)acetic acid composition with acetic anhydride and a catalyst to produce a 1-(3-(trifluoromethyl)phenyl)propan-2-one composition; and (c) reductively aminating the 1-(3-(trifluoromethyl)phenyl)propan-2-one composition with ethylamine using a borohydride reducing agent to produce a fenfluramine composition. Also provided are compositions and pharmaceutical ingredients prepared according to the subject methods including a pharmaceutically acceptable salt of fenfluramine and having less than 0.2% by weight in total of trifluoromethyl regioisomers.

Fenfluramine is an amphetamine drug that was once widely prescribed asan appetite suppressant to treat obesity. Fenfluramine is devoid of thepsychomotor stimulant and abuse potential of D-amphetamine and interactswith the 5-hydroxytryptamine (serotonin, 5-HT) receptors to release 5-HTfrom neurons. Fenfluramine has been investigated as havinganticonvulsive activity in the treatment of Dravet Syndrome, or severemyoclonic epilepsy in infancy, a rare and malignant epileptic syndrome.This type of epilepsy has an early onset in previously healthy children.

Anorectic treatment with fenfluramine has been associated with thedevelopment of cardiac valvulopathy and pulmonary hypertension,including the condition cardiac fibrosis which led to the withdrawal offenfluramine from world-wide markets. Interaction of fenfluramine'smajor metabolite norfenfluramine with the 5-HT2B receptor is associatedwith heart valve hypertrophy. In the treatment of epilepsy, the knowncardiovascular risks of fenfluramine are weighed against beneficialanticonvulsive activity.

SUMMARY

The present disclosure provides methods of preparing a fenfluramineactive pharmaceutical ingredient. Aspects of the subject methods includehydrolyzing a 2-(3-(trifluoromethyl)phenyl)acetonitrile composition toproduce a 2-(3-(trifluoromethyl)phenyl)acetic acid composition; reactingthe 2-(3-(trifluoromethyl)phenyl)acetic acid composition with aceticanhydride and a catalyst to produce a1-(3-(trifluoromethyl)phenyl)propan-2-one composition; and reductivelyaminating the 1-(3-(trifluoromethyl)phenyl)propan-2-one composition withethylamine using a borohydride reducing agent to produce a fenfluraminecomposition.

Also provided are fenfluramine compositions and pharmaceuticalingredients produced according to the subject methods that include areduced amount of one or more minor components such as impurities orreaction side products. In some cases, the compositions include apharmaceutically acceptable salt of fenfluramine having less than 0.2%by weight in total of trifluoromethyl regioisomers. Also provided arepharmaceutical compositions including the subject fenfluraminecompositions.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the metabolism-resistant fenfluramine analogs and methods ofusing the same as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is best understood from the following detaileddescription when read in conjunction with the accompanying drawings. Itis emphasized that, according to common practice, the various featuresof the drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1 illustrates the contributions of various precursor materials tothe structure of fenfluramine (1) in an exemplary retrosyntheticanalysis to acid (4).

FIG. 2 illustrates an exemplary HPLC chromatogram of a crude preparationof fenfluramine hydrochloride (210 nm UV absorbance).

FIG. 3 illustrates an exemplary HPLC chromatogram of a crystallizedfenfluramine hydrochloride composition (210 nm UV absorbance).

FIG. 4 illustrates a variety of synthetic pathways for preparation ofketone (2). An exemplary method that finds use in the subject methods ispreparation of ketone (2) from nitrile (5) via acid (4).

FIG. 5 illustrates a route to prepare ketone (2) from an Aryl Nitrostarting material via a diazonium intermediate. The diazonium route hasa disadvantage due to the potential formation of genotoxic intermediatesshown as boxed compounds (e.g., N-hydroxyaryl, N-nitrosamine and Nitrocompound).

DEFINITIONS

As used herein, the term “subject” refers to a mammal. Exemplary mammalsinclude, but are not limited to, humans, domestic animals (e.g., a dog,cat, or the like), farm animals (e.g., a cow, a sheep, a pig, a horse,or the like) or laboratory animals (e.g., a monkey, a rat, a mouse, arabbit, a guinea pig, or the like). In certain embodiments, the subjectis human. “Patient” refers to human and non-human subjects, especiallymammalian subjects.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. As used herein, the terms “treating,”“treatment,” “therapeutic,” or “therapy” do not necessarily mean totalcure or abolition of the disease or condition. Any alleviation of anyundesired signs or symptoms of a disease or condition, to any extent canbe considered treatment and/or therapy. Furthermore, treatment mayinclude acts that may worsen the patient's overall feeling of well-beingor appearance. “Treatment,” as used herein, covers any treatment of adisease in a mammal, in some cases in a human, and includes: (a)preventing the disease or medical condition from occurring, such as,prophylactic treatment of a subject; (b) ameliorating the disease ormedical condition, such as, eliminating or causing regression of thedisease or medical condition in a patient; (c) suppressing the diseaseor medical condition, for example by, slowing or arresting thedevelopment of the disease or medical condition in a patient; or (d)alleviating a symptom of the disease or medical condition in a patient.

As used herein, the term pKa refers to the negative logarithm (p) of theacid dissociation constant (Ka) of an acid, and is equal to the pH valueat which equal concentrations of the acid and its conjugate base formare present in solution.

The term “salt” refers to an ionic compound that result from theneutralization reaction of an acid and a base, and is composed of atleast one cation (positively charged ion) and at least one anion(negative ion). In some embodiments, a salt is electrically neutral(without a net charge). Where applicable, the salt is a pharmaceuticallyacceptable salt, although this is not required for salts of intermediatecompounds that are not intended for administration to a patient. By wayof example, salts of the present compounds include those wherein thebasic compound is protonated by an inorganic or organic acid to form aconjugate acid cation, with the conjugate base of the inorganic ororganic acid as the anionic component of the salt. Salts of interestinclude, but are not limited to, hydrochloride salts. It is understoodthat for any of the structures depicted herein, such structures may alsoinclude any convenient salt forms.

The term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inmammals, such as humans.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (salts withcounterions having acceptable mammalian safety for a given dosageregime). Such salts can be derived from pharmaceutically acceptableinorganic or organic bases and from pharmaceutically acceptableinorganic or organic acids. “Pharmaceutically acceptable salt” refers topharmaceutically acceptable salts of a compound, which salts are derivedfrom a variety of organic and inorganic counter ions well known in theart and include, by way of example only, sodium, and the like; and whenthe molecule contains a basic functionality, salts of organic orinorganic acids, such as hydrochloride, and the like. Pharmaceuticallyacceptable salts of interest include, but are not limited to,hydrochloride salts.

The term “active pharmaceutical ingredient” (API) refers to a substanceor mixture of substances intended to be used in the manufacture of adrug product and that, when used in the production of a drug, becomes anactive ingredient in the drug product. Such substances are intended tofurnish pharmacological activity or other direct effect in thediagnosis, cure, mitigation, treatment or prevention of disease or toaffect the structure and function of the body.

“Solvate” refers to a complex formed by combination of solvent moleculeswith molecules or ions of the solute. The solvent can be an organiccompound, an inorganic compound, or a mixture of both. Some examples ofsolvents include, but are not limited to, methanol,N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.When the solvent is water, the solvate formed is a hydrate.

“Stereoisomer” and “stereoisomers” refer to compounds that have sameatomic connectivity but different atomic arrangement in space.Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers,and diastereomers.

“Tautomer” refers to alternate forms of a molecule that differ only inelectronic bonding of atoms and/or in the position of a proton, such asenol-keto and imine-enamine tautomers, or the tautomeric forms ofheteroaryl groups containing a —N═C(H)—NH— ring atom arrangement, suchas pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. Aperson of ordinary skill in the art would recognize that othertautomeric arrangements of the groups described herein are possible.

It will be appreciated that the term “or a salt or solvate orstereoisomer thereof” is intended to include all permutations of salts,solvates and stereoisomers, such as a solvate of a pharmaceuticallyacceptable salt of a stereoisomer of subject compound. It is understoodthat the term “or a salt thereof” is intended to include allpermutations of salts. It is understood that the term “or apharmaceutically acceptable salt thereof” is intended to include allpermutations of salts. It is understood that the term “or a solvatethereof” is intended to include all permutations of solvates. It isunderstood that the term “or a stereoisomer thereof” is intended toinclude all permutations of stereoisomers. It is understood that theterm “or a tautomer thereof” is intended to include all permutations oftautomers. Thus for example it follows that it is intended to include asolvate of a pharmaceutically acceptable salt of a tautomer of astereoisomer of subject compound.

“Pharmaceutically effective amount” and “therapeutically effectiveamount” refer to an amount of a compound sufficient to treat a specifieddisorder or disease or one or more of its symptoms and/or to prevent theoccurrence of the disease or disorder. In reference to tumorigenicproliferative disorders, a pharmaceutically or therapeutically effectiveamount comprises an amount sufficient to, among other things, cause thetumor to shrink or decrease the growth rate of the tumor.

The term “vehicle” refers to a diluent, adjuvant, excipient, or carrierwith which a compound of the invention is formulated for administrationto a mammal.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupercedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acompound” includes a plurality of such compounds and reference to “themethod” includes reference to one or more methods and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Before the present compounds and methods are described, it is to beunderstood that this invention is not limited to particular compoundsand methods described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

DETAILED DESCRIPTION

As summarized above, the present disclosure provides methods ofpreparing a fenfluramine active pharmaceutical ingredient. Aspects ofthe present disclosure include fenfluramine compositions andpharmaceutical ingredients produced according to the subject methodswhere particular undesirable minor components of interest aresubstantially eliminated from the composition. The subject methodsprovide for a combination of steps to produce a crude composition thatachieves a desirable minimum threshold for undesirable minor components,such as difficult to purify regioisomers, reaction side products andreagents. Active pharmaceutical ingredients for pharmaceuticalformulations are prepared via controlled and reproducible methods toachieve highly pure compositions of active agent which provide highlevels of safety, efficacy and quality in the resulting pharmaceuticalformulations. In some cases, impurities or undesirable minor componentsin pharmaceutical compositions can cause drug product instability, lossof potency and toxicity. The substantial elimination of such minorcomponents from the subject fenfluramine compositions provides acomposition that is suitable for use in pharmaceutical compositions asthe active pharmaceutical ingredient (API). The subject compositions canbe produced efficiently with a reduced need for purification,eliminating purification steps or improving the outcome of method stepssuch as those steps involving removal of difficult to removeregioisomers of fenfluramine.

The term “composition”, when used in the context of the subject methods,describes a material that is a starting material or a product of one ormore steps of the subject methods and which can contain a mixture ofcomponents. The composition can be referred to by its predominant ortarget component, e.g., a fenfluramine composition. In general terms, acomposition can include, in addition to a predominant target component,a mixture of other components, such as target isomers (e.g., astereoisomer or regioisomer), impurities, reaction side products,starting materials, carry-over components from previous steps, reagents,solvents, and the like. As used herein, the term “crude composition”refers to the material produced in the performance of a chemicalreaction procedure which has not been subjected to additionalpurification steps, e.g., separate post-reaction procedure steps, suchas chromatography or recrystallization steps. In the preparation of acrude composition, the material can be subjected to simple steps, e.g.,such as aqueous washes, solvent extractions and/or filtrations, whichare considered an integral part of the reaction procedure, because suchsteps are commonly used to terminate a chemical reaction and/or to“work-up” a reaction product. Such reaction workup steps are notconsidered to be additional purification steps, as described above, butare merely part of the preparation of a crude composition.

Methods of Preparation of Fenfluramine Compositions

Aspects of the subject methods include preparation of a fenfluraminecomposition from a 1-(3-(trifluoromethyl)phenyl)-propan-2-one precursorcomposition via reductive amination (Scheme 1).

Any convenient methods of reductive amination may be utilized to convertthe ketone (2) to fenfluramine (1) via an imine intermediate (la), e.g.,via a Schiff base formed between ethylamine (e.g., Et-NH₂) and theketone (2). Methods and reagents of interest include, but are notlimited to, those methods and reagents described by Abdel-Magid et al.(“Reductive Amination of Aldehydes and Ketones with SodiumTriacetoxyborohydride. Studies on Direct and Indirect ReductiveAmination Procedures”, J. Org. Chem., 1996, 61 (11), pp 3849-3862. Insome embodiments, the reductive amination reaction is performed underconditions that comprise contacting the1-(3-(trifluoromethyl)phenyl)propan-2-one composition with a solution of70% by weight of ethylamine/water and about 2.25 equivalents or more ofsodium triacetoxyborohydride dissolved in methanol as solvent. Incertain cases, the reaction (e.g., scheme 1) is performed at anindustrial scale (e.g., as described herein). In certain instances, theyield of the reaction (e.g., scheme 1) is 80% or more, such as 85% ormore, 90% or more, 95% or more, 98% or more or 99% or more.

Any convenient reducing agents can be used in the reductive aminationstep of the subject methods, e.g., to reduce the Schiff baseintermediate to the secondary amine product, fenfluramine. In someinstances, the reducing agent is a borohydride reducing agent. As usedherein, the term “borohydride reducing agent” is meant to include anyreducing agent that includes a BH⁻ group, such as any convenientborohydride, cyanoborohydride or triacetoxyborohydride reducing agenthaving the formula MBR₃H, where each R is independently H, alkyl, cyanoor acetoxy and M is a metal such as Na, Li or K. In some instances, thereducing agent is a cyanoborohydride reducing agent. In some instances,the reducing agent is a triacetoxyborohydride reducing agent. In somecases, the reducing agent is selected from sodium borohydride, sodiumcyanoborohydride, sodium triacetoxyborohydride, lithiumtriethylborohydride, nickel borohydride, potassium borohydride andcalcium borohydride. In certain instances the borohydride reducing agentis sodium triacetoxyborohydride (STAB; Na(CH₃COO)₃BH).

The 1-(3-(trifluoromethyl)phenyl)-propan-2-one (2) composition can beprepared from any convenient precursor composition. In some instances,the 1-(3-(trifluoromethyl)phenyl)-propan-2-one (2) composition isprepared from 2-(3-(trifluoromethyl)phenyl)acetic acid (4), e.g.,according to Scheme 2 via a Daikin-West reaction. As such, aspects ofthe subject methods include reacting the2-(3-(trifluoromethyl)phenyl)acetic acid composition with aceticanhydride and a catalyst to produce a1-(3-(trifluoromethyl)phenyl)propan-2-one composition.

The Daikin-west reaction provides for the conversion of an enolizablecarboxylic acid to a corresponding methyl ketone by reaction with anacetylation agent (e.g., acetic anhydride and a catalyst). In somecases, the catalyst is a nucleophilic catalyst. Any convenientnucleophilic catalyst can be used in junction with acetic anhydride inthe preparation of ketone (2) via Scheme 2. In some embodiments, thecatalyst is N-methylimidazole (i.e., 1-methylimidazole). The catalystand the acetic anhydride may combine to form an acetylating agent insitu. It is understood that a variety of other acetylating agents andprecursor reagents for producing an acetylation agent in situ may beutilized in the reaction step. In some cases, the method step includesaddition of a pre-formed acetylating agent directly to the acid (4).Methods and reagents of interest that find use in the preparation ofketone (2) include, but are not limited to, those described by Buchananin “The Dakin-West reaction”, Chem. Soc. Rev., 1988, 17, 91-109. In someembodiments, the reaction is performed under conditions that includecontacting the 2-(3-(trifluoromethyl)phenyl)acetic acid composition withabout 0.5 equivalents of 1-methylimidazole and about 5 equivalents ormore of acetic anhydride, optionally in a solvent. In certain instances,the yield of the reaction (e.g., scheme 2) is 80% or more, such as 85%or more, 90% or more, 95% or more, 98% or more or 99% or more.

The ketone (2) can be optionally purified before use in the stepoutlined in Scheme 1 using any convenient method. In some cases, theketone (2) is purified via formation of a bisulfite adduct. As usedhere, the terms “bisulfite adduct” and “bisulfite addition compound” areused interchangeably to refer to the product of addition of a bisulfiteion to a ketone compound. The bisulfite adduct of ketone (2) can be asolid which provides for a more facile removal of impurities from theadduct composition than is possible from the corresponding parent ketonecomposition.

Aspects of the subject methods include a combination of the individualsteps described herein, e.g., a combination of steps as step forth inScheme 4. Before or after any of the steps described an optionaladditional purification step (e.g., crystallization step) may beperformed. In some embodiments, the method includes reacting a2-(3-(trifluoromethyl)phenyl)acetic acid composition with aceticanhydride and a catalyst to produce a1-(3-(trifluoromethyl)phenyl)propan-2-one composition; and reductivelyaminating the 1-(3-(trifluoromethyl)phenyl)propan-2-one composition withethylamine using a borohydride reducing agent to produce a fenfluraminecomposition.

The 2-(3-(trifluoromethyl)phenyl)acetic acid (4) composition can beprepared from any convenient precursor composition. In some instances,the 2-(3-(trifluoromethyl)phenyl)acetic acid composition is preparedfrom a 2-(3-(trifluoromethyl)phenyl)acetonitrile composition, e.g.,according to the reaction of Scheme 5. As such, aspects of the subjectmethod includes hydrolyzing a 2-(3-(trifluoromethyl)phenyl)acetonitrile(5) composition to produce a 2-(3-(trifluoromethyl)phenyl)acetic acid(4) composition.

Hydrolysis of the nitrile (5) to the acid (4) can be achieved using anyconvenient methods. In some cases, the hydrolysis of the nitrile (5) isachieved via acid-catalyzed hydrolysis. In certain instances, thehydrolysis of the nitrile (5) is achieved via base-catalyzed hydrolysis.Hydrolysis may proceed via an amide intermediate (4a) under aqueousacidic conditions. In some embodiments of the method, hydrolysis of thenitrile (5) to the acid (4) is performed under aqueous acidicconditions. In certain instances, the yield of the reaction (e.g.,scheme 5) is 80% or more, such as 85% or more, 90% or more, 95% or more,98% or more or 99% or more.

In some cases, the method includes hydrolyzing a2-(3-(trifluoromethyl)phenyl)acetonitrile (5) composition to produce a2-(3-(trifluoromethyl)phenyl)acetic acid (4) composition; and reacting a2-(3-(trifluoromethyl)phenyl)acetic acid (4) composition with aceticanhydride and a catalyst to produce a1-(3-(trifluoromethyl)phenyl)propan-2-one (2) composition (see e.g.,Scheme 6).

Aspects of the subject methods include a combination of the stepsdescribed herein e.g., a combination of steps as described in Scheme 7.Before or after any of the steps described, an optional additionalpurification step (e.g., crystallization step) may be performed. In someembodiments, the method includes hydrolyzing a2-(3-(trifluoromethyl)phenyl)acetonitrile (5) composition to produce a2-(3-(trifluoromethyl)phenyl)acetic acid (4) composition; reacting the2-(3-(trifluoromethyl)phenyl)acetic acid (4) composition with aceticanhydride and a catalyst to produce a1-(3-(trifluoromethyl)phenyl)propan-2-one (2) composition; andreductively aminating the 1-(3-(trifluoromethyl)phenyl)propan-2-one (2)composition with ethylamine using a borohydride reducing agent toproduce a fenfluramine (1) composition.

In some embodiments of the method, the fenfluramine composition (e.g., acrude fenfluramine composition) that is produced has the followingprofile: 80% or more by weight of fenfluramine or a salt thereof, suchas 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99%or more, 99.5% or more, or even more by weight of the fenfluramine orsalt thereof; 1% or less by weight of 2-fenfluramine regioisomer or asalt thereof, such as 0.5% or less, 0.2% or less, or 0.1% or less, 0.05%or less, 0.01% or less, or even less by weight of 2-fenfluramineregioisomer or a salt thereof; 1% or less by weight of 4-fenfluramineregioisomer or a salt thereof, such as 0.5% or less, 0.2% or less, or0.1% or less, 0.05% or less, 0.01% or less, or even less by weight of4-fenfluramine regioisomer or a salt thereof; and 10% or less by weightof fenfluramine reduced alcohol side product, such as 5% or less, 2% orless, or 1% or less, 0.5% or less, 0.1% or less by weight offenfluramine reduced alcohol side product.

In some embodiments, the method is a method of preparing fenfluraminefree base. As such, the fenfluramine composition can includefenfluramine free base. Fenfluramine free base that is preparedaccording to the subject methods may be converted to any convenient saltform, e.g., a salt of the conjugate acid of the secondary amino group offenfluramine (fenfluramine.H⁺X⁻), using a variety of methods. Theformation of a fenfluramine salt can be performed as part of thereductive amination step of Scheme 1 (e.g., in situ), or salt formationcan be performed in an optional subsequent step. In some cases, the saltform is a pharmaceutically acceptable salt of fenfluramine Salts ofinterest include, but are not limited to, a hydrochloride salt. Incertain instances, the pharmaceutically acceptable salt form offenfluramine is a hydrochloride salt.

The subject methods provide for substantial elimination of one or moreundesirable minor components from the crude fenfluramine composition orfenfluramine salt composition, such that final additional purificationsteps can be achieved easily with high efficiency and/or high yield toproduce a high quality active pharmaceutical composition.

One or more additional purification steps may be performed on the crudefenfluramine composition (e.g., that includes a free base or a salt formof fenfluramine) prepared according to the subject methods. In certaininstances, the purification step includes crystallization offenfluramine or the salt form of fenfluramine from the crudecomposition. The crystalline fenfluramine salt form can have a desirablepolymorphism, high crystallinity, water solubility and/or stability. Insome cases, the subject methods provide for a crystalline fenfluraminehydrochloride salt that is a single polymorph that is free-flowing,non-hygroscopic and having a high melting temperature.

In some embodiments of the method, the composition produced comprises apharmaceutically acceptable salt of fenfluramine and has followingpurity profile: 90% or more of the pharmaceutically acceptable salt offenfluramine, such as 95% or more, 96% or more, 97% or more, 98% ormore, 99% or more, 99.5% or more, 99.8% or more, 99.9% or more, or evenmore by weight of the pharmaceutically acceptable salt of fenfluramine;1% or less by weight of 2-fenfluramine; 1% or less by weight of2-fenfluramine regioisomer or a salt thereof, such as 0.5% or less, 0.2%or less, or 0.1% or less, 0.05% or less, 0.01% or less, or even less byweight of 2-fenfluramine regioisomer or a salt thereof; 1% or less byweight of 4-fenfluramine regioisomer or a salt thereof, such as 0.5% orless, 0.2% or less, or 0.1% or less, 0.05% or less, 0.01% or less, oreven less by weight of 4-fenfluramine regioisomer or a salt thereof; and5% or less by weight of fenfluramine reduced alcohol side product, suchas 3% or less, 2% or less, or 1% or less, 0.5% or less, 0.1% or less byweight of fenfluramine reduced alcohol side product. In certainembodiments, the composition produced according to the subject methodsis a fenfluramine active pharmaceutical ingredient comprising apharmaceutically acceptable salt of fenfluramine and having 0.2% or lessby weight in total of trifluoromethyl regioisomers, such as 0.1% orless, 0.05% or less, 0.03% or less, 0.01% or less, or even less byweight of trifluoromethyl regioisomers. In certain embodiments, thefenfluramine active pharmaceutical ingredient has a purity profilecomprising: at least 90% (e.g., at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, atleast 99.9%, or more) by weight of a pharmaceutically acceptable salt offenfluramine; less than 0.2% by weight (e.g., less than 0.1%, less than0.05%, less than 0.03%, less than 0.01% by weight) of 2-fenfluramine;less than 0.2% by weight (e.g., less than 0.1%, less than 0.05%, lessthan 0.03%, less than 0.01% by weight) of 4-fenfluramine; and less than1% by weight (e.g., less than 0.5%, less than 0.3%, less than 0.1%, lessthan 0.05% by weight) of fenfluramine alcohol.

The subject methods provide for the preparation of a racemic mixture ofenantiomers of fenfluramine. The enantiomers of fenfluramine may bereferred to as: dexfenfluramine (i.e.,(S)—N-ethyl-1-[3-(trifluoromethyl)phenyl]-propan-2-amine,(+)-fenfluramine or (S)-fenfluramine); and levofenfluramine (i.e.,(2R)—N-ethyl-1-[3-(trifluoromethyl)phenyl]-2-propanamine,(−)-fenfluramine or (R)-fenfluramine). The fenfluramine enantiomers orsalts thereof can be separated from each other using any convenientmethods. Methods of interest for separation and purification offenfluramine enantiomers include, but are not limited to, chiralresolution by crystallization and chiral column chromatography. As such,in some embodiments, the method further includes performing a chiralseparation of a racemic fenfluramine composition, or a salt thereof, toproduce a non-racemic fenfluramine composition comprising a predominantstereoisomer of fenfluramine By non-racemic is meant a compositionhaving an enantiomeric excess of at least 50%, such as at least 60%, atleast 70%, at least 80%, at least 90%, at least 95% or at least 99% ofone stereoisomer, e.g., a predominant stereoisomer. As used herein, theterm “predominant stereoisomer” is meant to encompass a compositionincluding only one stereoisomer or a composition that includesstereoisomer mixtures.

In some cases, the active pharmaceutical ingredient composition is anon-racemic composition including (S)-fenfluramine or a pharmaceuticallyacceptable salt thereof as the predominant stereoisomer. In some cases,the active pharmaceutical ingredient composition is a non-racemiccomposition including (R)-fenfluramine or a pharmaceutically acceptablesalt thereof as the predominant stereoisomer. In some cases, thenon-racemic composition that is produced includes only one stereoisomer.

Minor Components

As summarized above, the compositions of the subject methods, e.g.,starting material compositions, intermediate compositions and finalfenfluramine compositions may provide for the substantial elimination ofone or more minor components, which is achieved by the subject methodsto produce compositions that find use as an active pharmaceuticalingredient (API), or precursor thereof, for pharmaceutical compositions.The subject methods provide for the substantial elimination ofundesirable minor components is a variety of ways. As used herein, by“substantially eliminate” is meant the achievement of a desirableminimum threshold for a minor component of interest, such that the minorcomponent, if present, is present at a level at or below the threshold.As used herein, the term “substantially devoid” refers to a compositionwhere a minor component of interest is either not present or is presentat a level at or below the minimum threshold. The desirable minimumthreshold for a minor component of interest may vary according to thenature of the component and whether the composition in an intermediatecomposition or a fenfluramine composition of interest. In someinstances, the desirable minimum threshold of a minor component ofinterest that is achieved is 10% by weight or less, such as 5% or less,4% or less, or 3% or less, 2% or less, 1% or less 0.5% or less, 0.4% orless, 0.3% or less, 0.2% or less, 0.15% or less 0.1% or less, 0.09% orless, 0.08% or less, 0.07% or less, 0.06% or less, 0.05% or less, 0.03%or less, or 0.01% or less. In certain instances, the minor component ofinterest is completely eliminated from the compositions of interest,i.e., the composition is devoid of the minor component (e.g., is notdetected or is below the detectable limit of the component).

In some cases, the particular combination of steps utilized in thesubject method works to eliminate a minor component of interest. Incertain instances, purification of an intermediate composition, e.g.,via crystallization, achieves substantial elimination of a minorcomponent that would be difficult to remove if the minor component,e.g., a regioisomer, was carried forward to a later step in thesynthesis. In certain instances, the performance of a particular methodstep provides for a selectivity of reaction, whereby a minor componentof interest is not transformed by the reaction conditions as the majorcomponent is, and thus may be more easily removed, e.g., as aregioisomer of the starting material rather than the product of areaction or particular method step. In some cases, the particularcombination of steps utilized in the subject methods avoids the use ofone or more chemical reagents, solvents and/or reactants that isrequired via conventional methods and which are lead to undesirableminor components in the product compositions. Minor components ofinterest which may be substantially eliminated include but are notlimited to, product isomers, side products, aldehydes, ketones,peroxides, metals (e.g., heavy metal and metal catalysts),nitrate/nitrite, trace solvents, and organic acids. Various minorcomponents and details of their substantial elimination from the subjectcompositions are now described in greater below. Minor components ofinterest that may be substantially eliminated according to the subjectmethods include any impurities, by-products, starting materials andminor components described herein, including but not limited to, acetateimpurity, dimer impurity, Acetamide impurity,1-((3-trifluoromethyl)phenyl)acetone, fenfluramine regioisomers,Fenfluramine Alcohol, N-(3-(trifluoromethyl)-benzyl)ethanamine,norfenfluramine and any one of the impurities of Table 7.

Regioisomers

In some instances, a regioisomer of fenfluramine, or a precursorthereof, can be present as a minor component of any one of the subjectcompositions that find use in the subject methods. Fenfluramine andsynthetic precursors thereof can include a 3-trifluoromethyl substitutedphenyl group. As used here, the terms, “trifluoromethyl regioisomer” and“trifluoromethyl-phenyl regioisomer” are used interchangeably to referto an isomer(s) of fenfluramine, or any one of the synthetic precursorsdescribed herein, where the trifluoromethyl substituent is located ateither the 2-position or the 4-position of the substituted phenyl ringrather than at the 3-position corresponding to fenfluramine. As such,the terms “2-trifluoromethyl regioisomer” and “4-trifluoromethylregioisomer” can be used herein to describe particular minor componentsof any intermediate composition or final composition that finds use inthe subject methods.

The 2-(3-(trifluoromethyl)phenyl)acetonitrile composition startingmaterial of the subject methods can include regioisomers. In some cases,the regioisomers derive from the method of preparation of the2-(3-(trifluoromethyl)phenyl)acetonitrile from trifluoromethyl benzene.In some instances, the 2-(3-(trifluoromethyl)phenyl)acetonitrilecomposition comprises at least 0.2% by weight, such as at least 0.3%, atleast 0.4%, at least 0.5%, at least 1.0%, at least 1.5%, at least 2%, atleast 3%, at least 4%, at least 5%, at least 10%, or even more by weightof trifluoromethyl-phenyl regioisomers (e.g., a combined total of2-(2-(trifluoromethyl)phenyl)acetonitrile and2-(4-(trifluoromethyl)phenyl)acetonitrile). In some instances, the2-(3-(trifluoromethyl)phenyl)acetonitrile composition comprises at least0.2% by weight, such as at least 0.3%, at least 0.4%, at least 0.5%, atleast 1.0%, at least 1.5%, at least 2%, at least 3%, at least 4%, atleast 5%, at least 10%, or even more by weight of2-(4-(trifluoromethyl)phenyl)acetonitrile). In some instances, the2-(3-(trifluoromethyl)phenyl)acetonitrile composition comprises at least0.2% by weight, such as at least 0.3%, at least 0.4%, at least 0.5%, atleast 1.0%, at least 1.5%, at least 2%, at least 3%, at least 4%, atleast 5%, at least 10%, or even more by weight of2-(2-(trifluoromethyl)phenyl)acetonitrile). In certain instances, the2-(3-(trifluoromethyl)phenyl)acetonitrile composition includes minorregioisomer components that are carried over to the next composition,e.g., a 2-(3-(trifluoromethyl)phenyl)acetic acid composition. As such,the 2-(3-(trifluoromethyl)phenyl)acetic acid composition produced as anintermediate in the subject method can also include regioisomers (e.g.,2-(2-(trifluoromethyl)phenyl)acetic acid and2-(4-(trifluoromethyl)phenyl)acetic acid) at the same levels as aredescribed herein for the 2-(3-(trifluoromethyl)phenyl)acetonitrilecomposition starting material.

The subject methods provide for removal of 2- and/or 4-regioisomers asminor components of an intermediate composition in various ways. In someembodiments, the method includes purifying the2-(3-(trifluoromethyl)phenyl)acetic acid composition to produce acomposition substantially devoid of one or both of thetrifluoromethyl-phenyl regioisomers. In certain instances, thecomposition is also substantially devoid of benzaldehyde that is presentin the acetonitrile starting material. In certain instances, thecomposition is also substantially devoid of trifluoromethyl-benzaldehydethat is present in the acetonitrile starting material. In certaininstances, purifying the 2-(3-(trifluoromethyl)phenyl)acetic acidcomposition to remove a portion or all of the minor regioisomercomponents can be achieved via crystallization of the2-(3-(trifluoromethyl)phenyl)acetic acid. As used herein, the term“substantially devoid of a trifluoromethyl-phenyl regioisomer” meansless than 0.5% by weight, such as less than 0.4%, less than 0.3%, lessthan 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than0.07%, less than 0.06%, less than 0.05%, less than 0.03%, or even less.Any convenient methods of crystallization or recrystallization can beutilized in the subject methods.

After purification, e.g., crystallization, of the2-(3-(trifluoromethyl)phenyl)acetic acid composition, the compositioncan include less than 0.5% by weight, such as less than 0.4%, less than0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%,less than 0.07%, less than 0.06%, less than 0.05%, less than 0.03%, oreven less of 2-(2-(trifluoromethyl)phenyl)acetic acid. Afterpurification, e.g., crystallization, of the2-(3-(trifluoromethyl)phenyl)acetic acid composition, the compositioncan include less than 0.5% by weight, such as less than 0.4%, less than0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%,less than 0.07%, less than 0.06%, less than 0.05%, less than 0.03%, oreven less of 2-(4-(trifluoromethyl)phenyl)acetic acid. Afterpurification, e.g., crystallization, of the2-(3-(trifluoromethyl)phenyl)acetic acid composition, the compositioncan include less than 0.5% by weight, such as less than 0.4%, less than0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%,less than 0.07%, less than 0.06%, less than 0.05%, less than 0.03%, oreven less of benzaldehyde.

In some embodiments, the method includes reacting the2-(3-(trifluoromethyl)phenyl)acetic acid composition with aceticanhydride and a catalyst to produce a1-(3-(trifluoromethyl)phenyl)propan-2-one composition, where the2-(3-(trifluoromethyl)phenyl)acetic acid is selectively converted to theketone in the presence of unreacted 2-(2-(trifluoromethyl)phenyl)aceticacid. The subject method provides for facile removal of 2-regioisomerthat is present because this regioisomer is not carried through thereaction at the same rate as the target 3-trifluoromethyl compound. Insome cases, the method further comprises removing unreacted2-(2-(trifluoromethyl)phenyl)acetic acid regioisomer from the1-(3-(trifluoromethyl)phenyl)propan-2-one composition. As such, in someinstances, the crude 1-(3-(trifluoromethyl)phenyl)propan-2-onecomposition is substantially devoid (e.g., includes less than 0.5% byweight, such as less than 0.4%, less than 0.3%, less than 0.2%, lessthan 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than0.06%, less than 0.05%, less than 0.03%, or even less) of 2-regioisomerof the ketone product.

Removal of regioisomer minor components present in the acetonitrilestarting material may be achieved in stages during performance of thesynthetic method. In some embodiments, a first portion of theregioisomer minor components present in the starting material areremoved from the 2-(3-(trifluoromethyl)phenyl)acetic acid composition,e.g., via crystallization. In certain instance, a second portion of theregioisomer minor components present that are carried throughintermediate compositions of the subject method are removed viaselective reaction of the 2-(3-(trifluoromethyl)phenyl)acetic acid,e.g., as described herein. In certain instances, a third portion of theregioisomer minor components present that are carried throughintermediate compositions of the subject method are removed viapurification of a fenfluramine composition.

Benzaldehyde and Trifluorobenzaldehyde

Depending on the method of preparation of the2-(3-(trifluoromethyl)phenyl)acetonitrile, the starting materialcomposition can include benzaldehyde or trifluorobenzaldehyde as a minorcomponent. It is undesirable to have such a minor component present in apharmaceutical active ingredient. In some instances, the2-(3-(trifluoromethyl)phenyl)acetonitrile composition comprises at least0.2% by weight, such as at least 0.3%, at least 0.4%, at least 0.5%, atleast 1.0%, at least 2%, at least 5%, at least 10%, or even more byweight of benzaldehyde or trifluorobenzaldehyde as a minor component. Insome instances, any benzaldehyde or trifluorobenzaldehyde that ispresent as a minor component is substantially removed duringpurification, e.g., crystallization, of the2-(3-(trifluoromethyl)phenyl)acetic acid from the composition asdescribed herein. In certain instances, benzaldehyde is not present inthe 2-(3-(trifluoromethyl)phenyl)-acetonitrile starting materialcomposition due to its method of preparation.

Method of Preparation of Ketone (2)

The subject methods can include a particular combination of steps forpreparation of the ketone (2) that provide for one or more advantagesover other possible methods. FIG. 4 illustrates a variety of syntheticpathways that could be used for preparation of ketone (2). In certaincases, the particular method that finds use in the subject methods ispreparation of ketone (2) from nitrile (5) via acid (4).

In the subject methods, minor components (e.g., acetate and dimerimpurities) formed during the Dakin-West reaction (e.g., as described inScheme 2) can be subsequently substantially eliminated. In certaincases, these minor components are removed using a distillationprocedure. In certain instances, these minor components are removed viaa procedure including isolation of the product ketone (2) as thebisulfite salt (e.g., as described herein). The acetate and dimerimpurities are shown below.

In some instances, use of the bisulfite isolation procedure improves thepurity of the ketone by a factor of at least 30% (e.g., at least 40%, atleast 50%, or more) by removing these and other impurities. In someembodiments, the subject methods provide for substantial elimination ofthe acetate impurity from the ketone (2) composition. In someembodiments, the subject methods provide for substantial elimination ofthe dimer impurity from the ketone (2) composition.

FIG. 5 illustrates a diazonium route to prepare ketone (2) from an ArylNitro starting material. The diazonium route has a disadvantage due tothe potential formation of genotoxic intermediates shown as boxedcompounds (e.g., N-hydroxyaryl, N-nitrosamine and Nitro compound). Insome cases, removal of such impurities and/or demonstrating theirabsence is costly and time consuming and sometimes difficult to achievetechnically. Aspects of the subject methods include a synthetic routethat substantially eliminates the undesirable minor components that arepossible via the route shown in FIG. 5, thereby circumventing thepotential for such toxic and/or undesirable compounds to be present inthe subject compositions.

In some cases, the subject methods provide for elimination of isomer(e.g., a regioisomer) by-products of the 3-trifluoroaniline startingmaterial described in FIG. 5. Such by-products can be present in3-trifluoroaniline compositions, carried through synthetic steps, and bedifficult to substantially eliminate from downstream compositions. Insome instances of the subject methods, crystallization of the Acid (4)resulting from hydrolysis of the nitrile (5) provides crystalline Acid(4) which provides for a facile removal of such isomers early insynthesis. Removing impurities and/or undesirable isomers early in asynthesis can be preferred, especially if such impurities are carriedthrough to final product compositions, as purification of a finalproduct at the end of a synthesis is more costly (e.g., in losses ofvaluable product) and impacts cost of goods more greatly than removingsuch minor components early in synthesis before raw materials areinvested along the process.

Eliminated Toxic Reagents

The subject methods include a particular synthetic pathway andcombination of chemical reactions (e.g., as described above) thatprovides for the elimination of certain undesirable reagents and/orsolvents (e.g., Class 1 or Class 2 solvents that have known or they havestrongly suspected carcinogenic activity and/or are environmentalhazards). Class 1 and 2 solvents of interest which can be eliminatedfrom the fenfluramine composition by practicing the subject methodsinclude, but are not limited to, any solvent listed on the InternationalConference on Harmonization (ICH) Q3C list and guidance for Industry(February 2012, Revision 2, US Dept. HHS), such as acetonitrile, benzeneand substituted benzenes, carbon tetrachloride, chloroform, cyclohexane,1,2-dichloroethane, 1,1-dichloroethane, 1,2-dimethoxyethane, DMF,1,4-dioxane, methanol, methylbutyl ketone, N-methylpyrrolidinone,pyridine, toluene, 1,1,1-trichloroethane, 1,1,2-trichloroethene, andxylene. The subject methods also provide for the elimination of avariety of undesirable and/or toxic reagents from the fenfluraminecomposition that is produced by practicing the subject methods. Forexample, by including a reductive amination step according to the methoddepicted in Scheme 1, alternative synthetic pathways that require use ofpotentially toxic metal catalysts are avoided. By eliminating the use ofsuch reagents and/or solvents from the synthetic pathway of the subjectmethods, potentially toxic minor components are eliminated from thefenfluramine composition. As such, the subject fenfluramine compositioncan be referred to as being substantially devoid of the minor componentof interest. In some instances, one or more potential heavy metalcomponents such as Pb, As, Cd, Hg, Pb, Co, Mo, Se and V aresubstantially eliminated. In certain instances, one or more Class 1solvents are substantially eliminated (e.g., below an acceptablethreshold limit as adopted under ICH Q3C). In certain instances, benzenesolvent is substantially eliminated, e.g., below a concentration limitof 2 ppm. In certain instances, carbon tetrachloride solvent issubstantially eliminated, e.g., below a concentration limit of 4 ppm. Incertain instances, 1,2-dichloroethane solvent is substantiallyeliminated, e.g., below a concentration limit of 5 ppm. In certaininstances, 1,2-dichloroethane solvent is substantially eliminated, e.g.,below a concentration limit of 8 ppm. In certain instances,1,1,1-trichloroethane solvent is substantially eliminated, e.g., below aconcentration limit of 1500 ppm. A minor component can be consideredcompletely eliminated from the subject compositions when thefenfluramine is produced via a method where the minor component is notused in any synthetic step or present in a starting material.

Fenfluramine Alcohol

As used herein, the terms “fenfluramine alcohol” and “reduced alcoholside product” are used interchangeable to refer to the product of ketonereduction to alcohol that can occur in the reductive amination step ofScheme 1, depicted below.

The subject methods provide for substantial elimination of fenfluraminealcohol from the subject compositions. In some instances, the crudefenfluramine composition has less than 10% by weight of the reducedalcohol side product, such as less than 9%, less than 8%, less than 7%,less than 6%, less than 5%, less than 4%, less than 3%, less than 2%,less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%,less than 0.1%, less than 0.05% or even less. In some instances, thecrude fenfluramine composition has 10% or less by weight of the reducedalcohol side product, such as 9% or less, 8% or less, 7% or less, 6% orless, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9%or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.2% orless, 0.1% or less, 0.05% or less, or even less.

Norfenfluramine

Norfenfluramine is a potential impurity of compositions that includefenfluramine. The subject methods provide for substantial elimination ofnorfenfluramine from the subject compositions. In some instances, thecrude fenfluramine composition has less than 10% by weight ofnorfenfluramine, such as less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, lessthan 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%,less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, lessthan 0.1%, less than 0.05% or even less. In some instances, the crudefenfluramine composition includes has 10% or less by weight ofnorfenfluramine, such as 9% or less, 8% or less, 7% or less, 6% or less,5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% orless, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% orless, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, or evenless.

Methods of Use

Fenfluramine and the fenfluramine compositions described herein may beemployed in a variety of methods. Aspects of the present disclosureinclude a method that includes administering to a subject in needthereof a therapeutically effective amount of a fenfluraminepharmaceutical composition (e.g., as described herein) to treat orprevent a disease or condition of interest. By “therapeuticallyeffective amount” is meant the concentration of a compound that issufficient to elicit the desired biological effect (e.g., treatment orprevention of epilepsy). Diseases and conditions of interest include,but are not limited to, epilepsy, a neurological related diseases,obesity and obesity related diseases.

In some embodiments, the subject method includes administering to asubject a subject composition to treat a neurological related disease.Neurological related diseases of interest include, but are not limitedto, epilepsy, and Dravet syndrome. In certain embodiments, the subjectis human. In certain instances, the subject suffers from Dravetsyndrome. In certain embodiments, the compound is administered as apharmaceutical preparation.

Thus, according to a still further aspect of the present disclosure,there is provided a method of stimulating one or more 5-HT receptors inthe brain of a patient by administering an effective dose of afenfluramine composition to said patient, said one or more 5-HTreceptors being selected from one or more of 5-HT₁, 5-HT_(1A),5-HT_(1B), 5-HT_(1C), 5-HT_(1D), 5-HT_(1E), 5-HT_(1F), 5-HT₂, 5-HT_(2A),5-HT_(2B), 5-HT_(2C), 5-HT₃, 5-HT₄, 5-HT₅, 5-HT_(5A), 5-HT_(5B), 5-HT₆,and 5-HT₇ amongst others. In some instances, the 5-HT receptor is5-HT_(2B). In certain embodiments of this aspect of the invention, thepatient has been diagnosed with Dravet Syndrome. In some instances, themethod is a method of treating Dravet Syndrome that includes ofstimulating one or more 5-HT receptors in the brain of a patient byadministering an effective dose of a fenfluramine composition to saidpatient, said one or more 5-HT receptors being selected from one or moreof 5-HT_(1D), 5-HT_(2A) and 5-HT_(2C), among others.

There are a number of genetic mutations that are indicative of DravetSyndrome. Mutations in the SCN1A (such as partial or total deletionmutations, truncating mutations and/or missense mutations e.g. in thevoltage or pore regions S4 to S6), SCN1 B (such as the region encodingthe sodium channel β1 subunit), SCN2A, SCN3A, SCN8A, SCN9A, GABRG2 (suchas the region encoding the γ2 subunit), GABRD (such as the regionencoding the δ subunit) and/or PCDH19 genes have been linked to DravetSyndrome.

Thus, according to a further aspect of the present invention, there isprovided a method of treating a patient that exhibits a mutation in one,some or all of the above genes by administering to that patient aneffective dose of a fenfluramine comp. In certain embodiments of thisaspect of the invention, the patient has been diagnosed with DravetSyndrome.

In embodiments of the invention, any effective dose of the fenfluraminecomposition can be employed. However, surprisingly low doses offenfluramine compositions are found by the inventors to be efficacious,particularly for inhibiting or eliminating seizures in epilepsypatients. Thus, in some cases, in a preferred embodiment of theinvention, a daily dose of less than about 10 mg/kg/day such as, lessthan about 9 mg/kg/day, less than about 8 mg/kg/day, less than about 7mg/kg/day, less than about 6 mg/kg/day, less than about 5 mg/kg/day,less than about 4 mg/kg/day, less than about 3 mg/kg/day, less thanabout 2 mg/kg/day, less than about 1 mg/kg/day, such as about 1.0mg/kg/day, about 0.9 mg/kg/day, about 0.8 mg/kg/day, about 0.7mg/kg/day, about 0.6 mg/kg/day, about 0.5 mg/kg/day, about 0.45mg/kg/day, about 0.4 mg/kg/day, about 0.3 mg/kg/day, about 0.25mg/kg/day or about 0.2 mg/kg/day to about 0.1 mg/kg/day, about 0.05mg/kg/day, or about 0.01 mg/kg/day is employed. Put differently, apreferred dose is less than about 10 mg/kg/day to about 0.01 mg/kg/day.In some cases, the dose is less than about 5 mg/kg/day to about 0.1mg/kg/day, such as less than about 5 mg/kg/day to about 0.5, mg/kg/day,less than about 4 mg/kg/day to about 0.5 mg/kg/day, less than about 3mg/kg/day to about 0.5 mg/kg/day, less than about 2 mg/kg/day to about0.5 mg/kg/day, or less than about 1.7 mg/kg/day to about 0.9 mg/kg/day.

As indicated above the dosing is based on the weight of the patient.However, for convenience the dosing amounts may be preset such as in theamount of 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, or 50mg. In certain instances, the dosing amount may be preset such as in theamount of about 0.25 mg to about 5 mg, such as about 0.5 mg, about 0.75mg, about 1.0 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2.0mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3.0 mg, about 3.25mg, about 3.5 mg, about 3.75 mg, about 4.0 mg, about 4.25 mg, about 4.5mg, about 4.75 mg, or about 5.0 mg. The dosing amounts described hereinmay be administered one or more times daily to provide for a dailydosing amount, such as once daily, twice daily, three times daily, orfour or more times daily, etc. In certain embodiments, the dosing amountis a daily dose of 30 mg or less, such as 30 mg, about 29 mg, about 28mg, about 27 mg, about 26 mg, about 25 mg, about 24 mg, about 23 mg,about 22 mg, about 21 mg, about 20 mg, about 19 mg, about 18 mg, about17 mg, about 16 mg, about 15 mg, about 14 mg, about 13 mg, about 12 mg,about 11 mg, about 10 mg, about 9 mg, about 8 mg, about 7 mg, about 6mg, about 5 mg, about 4 mg, about 3 mg, about 2 mg, or about 1 mg. Ingeneral the smallest dose which is effective should be used for theparticular patient. In some cases, the dose is generally well below thedosing used in weight loss.

Administration of the subject pharmaceutical compositions may besystemic or local. In certain embodiments, administration to a mammalwill result in systemic release of fenfluramine (for example, into thebloodstream). Methods of administration may include enteral routes, suchas oral, buccal, sublingual, and rectal; topical administration, such astransdermal and intradermal; and parenteral administration. Suitableparenteral routes include injection via a hypodermic needle or catheter,for example, intravenous, intramuscular, subcutaneous, intradermal,intraperitoneal, intraarterial, intraventricular, intrathecal, andintracameral injection and non-injection routes, such as intravaginalrectal, or nasal administration. In certain embodiments, thecompositions of the present disclosure are administered orally. Incertain embodiments, it may be desirable to administer one or morecompounds of the invention locally to the area in need of treatment.This may be achieved, for example, by local infusion during, topicalapplication, by injection, by means of a catheter, by means of asuppository, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers.

The dose of fenfluramine administered in the methods of the presentinvention can be formulated in any pharmaceutically acceptable dosageform including, but not limited to oral dosage forms such as tabletsincluding orally disintegrating tablets, capsules, lozenges, oralsolutions or syrups, oral emulsions, oral gels, oral films, buccalliquids, powder e.g. for suspension, and the like; injectable dosageforms; transdermal dosage forms such as transdermal patches, ointments,creams; inhaled dosage forms; and/or nasally, rectally, vaginallyadministered dosage forms. Such dosage forms can be formulated for oncea day administration, or for multiple daily administrations (e.g. 2, 3or 4 times a day administration).

In some embodiments, the subject method includes administering to asubject an appetite suppressing amount of the subject compound to treatobesity. Any of the methods of administration and dosage forms of thesubject compositions may be utilized in treating obesity.

Combination therapy includes administration of a single pharmaceuticaldosage formulation which contains the subject composition and one ormore additional agents; as well as administration of the subjectcomposition and one or more additional agent(s) in its own separatepharmaceutical dosage formulation. For example, a subject compositionand an additional agent active with appetite suppressing activity (e.g.,phentermine or topiramate) can be administered to the patient togetherin a single dosage composition such as a combined formulation, or eachagent can be administered in a separate dosage formulation. Whereseparate dosage formulations are used, the subject composition and oneor more additional agents can be administered concurrently, or atseparately staggered times, e.g., sequentially.

In some embodiments, the subject method is an in vitro method thatincludes contacting a sample with a subject composition. The protocolsthat may be employed in these methods are numerous, and include but arenot limited to, serotonin release assays from neuronal cells, cell-freeassays, binding assays (e.g., 5HT2B receptor binding assays); cellularassays in which a cellular phenotype is measured, e.g., gene expressionassays; and assays that involve a particular animal model for acondition of interest (e.g., Dravet syndrome).

Pharmaceutical Preparations

Also provided are pharmaceutical preparations that include fenfluramineactive pharmaceutical ingredient compositions prepared according to thesubject methods. Pharmaceutical preparations are compositions thatinclude a compound (either alone or in the presence of one or moreadditional active agents) present in a pharmaceutically acceptablevehicle. In some embodiments, the pharmaceutical composition includes afenfluramine composition (e.g., as described herein) formulated in apharmaceutically acceptable excipient.

The choice of excipient will be determined in part by the particularcompound, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of the pharmaceutical composition of the present invention.

The dosage form of fenfluramine employed in the methods of the presentinvention can be prepared by combining the fenfluramine composition withone or more pharmaceutically acceptable diluents, carriers, adjuvants,and the like in a manner known to those skilled in the art ofpharmaceutical formulation.

The subject compositions can be formulated into preparations forinjection by dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

In some embodiments, formulations suitable for oral administration caninclude (a) liquid solutions, such as an effective amount of thecompound dissolved in diluents, such as water, or saline; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient (fenfluramine), as solids or granules; (c) suspensions in anappropriate liquid; and (d) suitable emulsions. Tablet forms can includeone or more of lactose, mannitol, corn starch, potato starch,microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, moistening agents,preservatives, flavoring agents, and pharmacologically compatibleexcipients. Lozenge forms can include the active ingredient in a flavor,usually sucrose and acacia or tragacanth, as well as pastilles includingthe active ingredient in an inert base, such as gelatin and glycerin, orsucrose and acacia, emulsions, gels, and the like containing, inaddition to the active ingredient, such excipients as are describedherein.

The subject formulations can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They may alsobe formulated as pharmaceuticals for non-pressured preparations such asfor use in a nebulizer or an atomizer.

In some embodiments, formulations suitable for parenteral administrationinclude aqueous and non-aqueous, isotonic sterile injection solutions,which can contain anti-oxidants, buffers, bacteriostats, and solutesthat render the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. The formulations can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials, and can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid excipient, for example, water, forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions can be prepared from sterile powders, granules, andtablets of the kind previously described.

Formulations suitable for topical administration may be presented ascreams, gels, pastes, or foams, containing, in addition to the activeingredient, such carriers as are appropriate. In some embodiments thetopical formulation contains one or more components selected from astructuring agent, a thickener or gelling agent, and an emollient orlubricant. Frequently employed structuring agents include long chainalcohols, such as stearyl alcohol, and glyceryl ethers or esters andoligo(ethylene oxide) ethers or esters thereof. Thickeners and gellingagents include, for example, polymers of acrylic or methacrylic acid andesters thereof, polyacrylamides, and naturally occurring thickeners suchas agar, carrageenan, gelatin, and guar gum. Examples of emollientsinclude triglyceride esters, fatty acid esters and amides, waxes such asbeeswax, spermaceti, or carnauba wax, phospholipids such as lecithin,and sterols and fatty acid esters thereof. The topical formulations mayfurther include other components, e.g., astringents, fragrances,pigments, skin penetration enhancing agents, sunscreens (e.g.,sunblocking agents), etc.

For an oral pharmaceutical formulation, suitable excipients includepharmaceutical grades of carriers such as mannitol, lactose, glucose,sucrose, starch, cellulose, gelatin, magnesium stearate, sodiumsaccharine, and/or magnesium carbonate. For use in oral liquidformulations, the composition may be prepared as a solution, suspension,emulsion, or syrup, being supplied either in solid or liquid formsuitable for hydration in an aqueous carrier, such as, for example,aqueous saline, aqueous dextrose, glycerol, or ethanol, preferably wateror normal saline. If desired, the composition may also contain minoramounts of non-toxic auxiliary substances such as wetting agents,emulsifying agents, or buffers.

By way of illustration, the fenfluramine composition can be admixed withconventional pharmaceutically acceptable carriers and excipients (i.e.,vehicles) and used in the form of aqueous solutions, tablets, capsules,elixirs, suspensions, syrups, wafers, and the like. Such pharmaceuticalcompositions contain, in certain embodiments, from about 0.1% to about90% by weight of the active compound, and more generally from about 1%to about 30% by weight of the active compound. The pharmaceuticalcompositions may contain common carriers and excipients, such as cornstarch or gelatin, lactose, dextrose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, andalginic acid. Disintegrators commonly used in the formulations of thisinvention include croscarmellose, microcrystalline cellulose, cornstarch, sodium starch glycolate and alginic acid.

Particular formulations of the present disclosure are in a liquid form.The liquid may be a solution or suspension and may be an oral solutionor syrup which is included in a bottle with a pipette which is graduatedin terms of milligram amounts which will be obtained in a given volumeof solution. The liquid solution makes it possible to adjust thesolution for small children which can be administered anywhere from 0.5mg to 15 mg and any amount between in half milligram increments and thusadministered in 0.5, 1.0, 1.5, 2.0 mg, etc.

A liquid composition will generally consist of a suspension or solutionof the compound or pharmaceutically acceptable salt in a suitable liquidcarrier(s), for example, ethanol, glycerine, sorbitol, non-aqueoussolvent such as polyethylene glycol, oils or water, with a suspendingagent, preservative, surfactant, wetting agent, flavoring or coloringagent. Alternatively, a liquid formulation can be prepared from areconstitutable powder.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric. By “average” is meant thearithmetic mean. Standard abbreviations may be used, e.g., s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

General Synthetic Procedures

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any purificationprotocol known in the art, including chromatography, such as HPLC,preparative thin layer chromatography, flash column chromatography andion exchange chromatography. Any suitable stationary phase can be used,including normal and reversed phases as well as ionic resins. In certainembodiments, the disclosed compounds are purified via silica gel and/oralumina chromatography. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl,Springer-Verlag, New York, 1969.

During any of the processes for preparation of the subject compounds, itmay be necessary and/or desirable to protect sensitive or reactivegroups on any of the molecules concerned. This may be achieved by meansof conventional protecting groups as described in standard works, suchas J. F. W. McOmie, “Protective Groups in Organic Chemistry”, PlenumPress, London and New York 1973, in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer),Academic Press, London and New York 1981, in “Methoden der organischenChemie”, Houben-Weyl, 4^(th) edition, Vol. 15/1, Georg Thieme Verlag,Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide,Protein”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982,and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide andDerivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groupsmay be removed at a convenient subsequent stage using methods known fromthe art.

The subject compounds can be synthesized via a variety of differentsynthetic routes using commercially available starting materials and/orstarting materials prepared by conventional synthetic methods. A varietyof examples of synthetic routes that can be used to synthesize thecompounds disclosed herein are described in the schemes below.

Example 1

1. Fenfluramine Nomenclature & Structure

Chemical Abstract Service (CAS) Registry Number (RN): 404-82-0 (HClSalt), 458-24-2 (Parent Free Base)

Chemical Name: N-ethyl-α-methyl-3-(trifluoromethyl)-benzeneethanaminehydrochloride(1:1). Other Names: Fenfluramine HCl, DL-Fenfluramine,(±)-Fenfluramine

Structure of hydrochloride salt:

Stereochemistry: Fenfluramine HCl has one chiral center and is beingdeveloped as the racemate and contains dexfenfluramine andlevofenfluramine

Molecular Formula of hydrochloride salt: C12H16F3N.HCl

Molecular Mass/Weight: 267.72 g/mol

2. General Properties

Table 1 summarizes the chemical and physical properties of FenfluramineHCl.

TABLE 1 General Properties of Fenfluramine HCl Drug Substance PropertyResult Appearance (color, White to off-white powder physical form) DSC(melting 170° C. (melt/sublimation) point)^(a) TGA Onset 147° C. 0.03%at 150° C. 91% at 220° C. (evaporation) pKa (water) 10.15-10.38Solubility (mg/mL) Resultant pH 25° C. 37° C. Solubility pH 6.69 (water)54.13 71.22 (Aqueous) pH 1.73 buffer 25.34 53.68 pH 3.43 buffer 29.5061.97 pH 6.41 buffer 37.42 95.60 0.9% NaCl (water) 22.98 — SolventSolubility 25° C. (mg/mL) Solubility (Organic Ethanol 150 Solvents)Dichloromethane 30-35 Ethyl Acetate, 1-5 mg Tetrahydrofuran, Toluene,Acetonitrile UV Absorption Maxima: 210, 265 nm Solution pH (water) 6.69Hygroscopicity @30% RH: ~0.05% (Dynamic Vapor @60% RH: ~0.07% Sorption(DVS) @90% RH: ~0.20%^(a)) Polymorphism Fenfluramine HCl has beenconsistently isolated as a single crystalline Form 1 as determined byDSC and x-ray powder diffraction (XRPD) Solvation/Hydration FenfluramineHCl is isolated as a nonhydrated, nonsolvated solid Solution Stability 8weeks @ pH 6.7 phosphate buffer medium at 40° C. and 60° C. usingconcentrations of 0.5, 2.5 and 5.0 mg/ml. All conditions, no newimpurities >0.1% by HPLC. Solid Stability 8 weeks @ 40° C., 60° C. and80° C. 7 days at 150° C. All conditions, no new impurities >0.1% byHPLC.3. Synthesis of Fenfluramine Drug Substance

Scheme 3.1 shows a 2-step route of synthesis used to manufacture initialclinical supplies of Fenfluramine HCl from ketone (2). The batch size is4 kg performed in laboratory glassware (kilo lab). No chromatography isrequired and the process steps are amenable to scale-up. In process 1there is one isolated intermediate Fenfluramine Free Base (1) startingfrom commercially supplied 1-(3-(trifluoromethyl)phenyl) acetone (Ketone2). All steps are conducted under cGMPs starting from Ketone (2).

Scheme 3.2 shows a 4-step route of synthesis to Fenfluramine HCl thatcan be used for commercial supply. Route 2 utilizes the same 2-stepprocess used by Route 1 to convert Ketone (2) to Fenfluramine HCl withthe exception that Ketone (2) is synthesized under cGMP conditionsstarting from 3-(Trifluoromethyl)-phenyl acetic acid (Acid 4). BisulfateComplex (3) is an isolatable solid and can be purified beforedecomplexation to Ketone (2). In-situ intermediates which are oils areshown in brackets. Batch sizes of 10 Kg are performed. Commercial batchsizes of 20 kg are performed in fixed pilot plant equipment. Steps 1-2of Scheme 3.2 to manufacture Ketone (2) have been demonstrated on a 100g scale to provide high purity ketone (2) of >99.8% (GC & HPLC).Conversion of Ketone (2) to Fenfluramine using either Route 1 or 2 hasprovided similar purity profiles.

Starting materials are designated by enclosed boxes. Bracketed and nonbracketed compounds respectively indicate proposed in-situ and isolatedintermediates. NMI=N-Methyl Imidazole.4.1. Narrative Description (Route 1)

Step 1: Reductive Amination (Preparation of Fenfluramine Free Base 1)

A solution of ethylamine, water, methanol, and1-(3-(trifluoromethyl)phenyl) acetone (Ketone 2) was treated with sodiumtriacetoxyborohydride and stirred for 16 h at 25° C. at which time HPLCanalysis (IPC-1; In Process Control No. 1) showed the reaction to becomplete and sodium hydroxide solution was added until pH >10. Toluenewas added and the phases separated, and the aqueous phase (IPC-2) andorganic phase (IPC-3) are checked for remaining Fenfluramine andFenfluramine alcohol and the organic phase was reduced. Purified waterwas added and the pH adjusted to <2 using conc. HCl and the phases wereseparated. The aqueous phase was washed with toluene and the toluenephase (IPC-4) and the aqueous phase (IPC-5) was checked for Fenfluramineand Fenfluramine alcohol content. The aqueous phase containing productis pH adjusted to >10 using sodium hydroxide solution. The basic aqueousphase was extracted with MTBE until removal of Fenfluramine from theaqueous phase was observed by HPLC (<0.5 mg/ml) (IPC-6). The organicphase was dried over sodium sulfate and filtered. The filtrate wasconcentrated in vacuo to give the intermediate product Fenfluramine FreeBase 1 as a pale yellow oil tested per specifications described hereinwhich showed by NMR the material to contain 2.93% toluene giving anactive yield of 88.3% with a purity of 98.23% by HPLC (0.67%Fenfluramine alcohol).

Step 2: Salt Formation (Preparation of Fenfluramine HCl)

To a flask was charged ethanol and acetyl chloride. The solution wasstirred slowly overnight before ethyl acetate was added. The HCl inethyl acetate solution formed was polish filtered into a clean carboyand retained for later use. To a vessel was added Fenfluramine free base1 and MTBE. The Fenfluramine solution in MTBE was collected in twocarboys before the vessel was cleaned and checked for particulateresidue. The Fenfluramine solution was polish filtered into a vessel andcooled and HCl in ethyl acetate solution was added giving a final pH of6-7. The batch was stirred for 1 h and filtered. The product was driedunder vacuum at 40° C. The product (96.52% yield) was tested per IPC-7had a purity of 99.75% by HPLC and GC headspace analysis showed MTBE(800 ppm) and EtOAc (150 ppm) to be present. The product was then testedper specifications shown herein.

4.2. Narrative Description (Route 2)

Step 1: Preparation of Ketone Bisulfite Adduct

In Situ Intermediate

Procedure: Charge acetic anhydride, (2.8 vol, 3.0 wt, 5.0 eq.) to avessel and commence stirring. Cool the solution to −5 to 5° C.,targeting −4° C. Charge 1-methylimidazole, (0.2 vol, 0.21 wt, 0.5 eq.)to the mixture at −5 to 5° C. Caution: very exothermic. If necessary,adjust the temperature to 0 to 5° C. Charge ZX008 acid, (1.00 wt, 1.0eq.) to the mixture at 0 to 5° C. Caution: exothermic. Stir the mixtureat 0 to 5° C. until ≤2.1% area ZX008 acid by HPLC analysis, typically 7to 9 hours. Charge 15% w/w sodium chloride solution (2.0 vol) to themixture at 0 to 5° C., 60 to 90 minutes. Caution: very exothermic whichwill be slightly delayed. Warm the mixture to 18 to 23° C. over 45 to 60minutes and continue stirring for a further 30 to 45 minutes at 18 to23° C. Charge TBME, (5.0 vol, 3.7 wt) to the mixture and stir for 10 to15 minutes at 18 to 23° C. Separate the aqueous layer and retain theorganic layer. Back-extract the aqueous layer with TBME, (2×3.0 vol,2×2.2 wt) at 18 to 23° C. retaining each organic layer. Adjust the pH ofthe combined organic layer to pH 6.5 to 9.0, targeting 7.0 by charging20% w/w sodium hydroxide solution (5.3 to 8.3 vol) at 18 to 23° C.Caution: exothermic. Separate the aqueous layer and retain the organiclayer. Wash the organic layer with 4% w/w sodium hydrogen carbonatesolution (2×3.0 vol) at 18 to 23° C. Determine the residual ZX008 acidcontent in the organic layer by HPLC analysis, pass criterion ≤0.10%area ZX008 acid. Wash the organic layer with purified water, (2×3.0 vol)at 18 to 23° C. Concentrate the organic layer under reduced pressure toca. 2 vol at 40 to 45° C., targeting 43° C.

Determine the w/w assay of ZX008 ketone (WIP) in the mixture by 1H-NMRanalysis for information only and calculate the contained yield of ZX008ketone (WIP) in the mixture. Note: This step can be removed from theprocess since the process is robust and consistently delivers 80 to 90%th yield. The achieved yield was factored into the charges of thesubsequent steps.

Charge n-heptane, (4.0 vol, 2.7 wt) to the mixture at 40 to 45° C.,targeting 43° C. Concentrate the mixture to ca. 2 vol at 40 to 45° C.,targeting 43° C. Determine the TBME content in the mixture by 1H-NMRanalysis, (pass criterion ≤5.0% w/w TBME vs. ZX008 ketone). Chargen-heptane, (2.4 vol, 1.6 wt) at 40 to 45° C., targeting 43° C., vesselA. To vessel B, charge sodium metabisulfite, (0.82 wt, 0.88 eq.) at 18to 23° C. To vessel B, charge a solution of sodium hydrogen carbonate,(0.16 wt, 0.4 eq.) in purified water, code RM0120 (2.0 vol) at 18 to 23°C. followed by a line rinse with purified water, code RM0120 (0.4 vol)at 18 to 23° C. Caution: gas evolution. Heat the contents of vessel B to40 to 45° C., targeting 43° C. Charge the contents from vessel A tovessel B followed by a line rinse with n-heptane, (0.8 vol, 0.5 wt) at40 to 45° C., targeting 43° C. Stir the mixture for 1 to 1.5 hours at 40to 45° C., targeting 43° C. Charge n-heptane, code RM0174 (3.2 vol, 2.2wt) to the mixture with the temperature being allowed to cool to 18 to45° C. at the end of the addition. Cool the mixture to 18 to 23° C. atapproximately constant rate over 45 to 60 minutes. Stir the mixture at18 to 23° C. for 1.5 to 2 hours.

Sample the mixture to determine the residual ZX008 ketone content by1H-NMR analysis, (pass criterion ≤10.0% mol, target 5.0% mol ZX008ketone vs. ZX008 ketone bisulfite adduct). Filter the mixture and slurrywash the filter-cake with n-heptane, (2×2.0 vol, 2×1.4 wt) at 18 to 23°C. Dry the solid at up to 23° C. until the water content is ≤10.0% w/wwater by KF analysis according to AKX reagent. At least 16 hours.Determine the w/w assay of the isolated ZX008 ketone bisulfite adduct by1H-NMR analysis and calculate the contained yield of ZX008 ketonebisulfite adduct.

Yields and Profiles: The yield for the stage 1 Demonstration batch issummarized Table below. Input: 1700.0 g uncorr., acid, 99.50% area (QC,HPLC), 2-isomer not detected, 4-isomer 0.02% area, RRT1.58 (previouslynot observed) 0.48% area as per the preparative method. The analyticaldata is summarized in Table 1A below.

TABLE 1A Table for isolated yields for step 1 Demonstration batch Corr.% w/w % area Reference Corr. Yield (¹H- (HPLC, number Input Output (%th)** NMR)* QC) Comments Batch A1 1700.0 g 1500.1 g 89.1 45.0 -.- Crudeketone as TBME sol. Batch A2 1500.1 g 1716.1 77.8 76.0 98.15 Bisulfiteadduct only 67.3 Overall product

Step 2: Preparation of Ketone

Procedure: Charge toluene, (5.0 vol, 4.3 wt), and purified water, (5.0vol) to the vessel and commence stirring. If necessary, adjust thetemperature to 18 to 23° C. and charge ZX008 ketone bisulfite adduct,(1.00 wt corrected for % w/w assay) to the mixture at 18 to 23° C.Charge 20% w/w sodium hydroxide solution to the mixture at 18 to 23° C.adjusting the pH of the mixture to pH 8.0 to 12.0, targeting 9.0 (0.5 to1.0 vol).

Separate the lower aqueous layer and retain the top organic layer. Washthe organic layer with purified water, (3.0 vol) at 18 to 23° C.Concentrate the organic layer under reduced pressure to ca. 2 vol at 45to 50° C., targeting 48° C. Charge methanol, (5.0 vol, 4.0 wt) to themixture at 45 to 50° C., targeting 48° C. Re-concentrate the mixtureunder reduced pressure to ca. 2 vol at 45 to 50° C., targeting 48° C.Repeat steps 7 and 8 once before continuing with step 9. Cool themixture to 18 to 23° C. Clarify the mixture into a tared, suitably-sizeddrum followed by a methanol (1.0 vol, 0.8 wt) line rinse at 18 to 23° C.Determine the w/w assay of ZX008 ketone (WIP) in the mixture by 1H-NMRanalysis and calculate the contained yield of ZX008 ketone (WIP) in themixture. Determine the toluene content in the mixture by 1H-NMRanalysis.

Yields and Profiles: The yield for the step 2 Demonstration batch issummarized in Table 1B below. Input: 1200.0 g corr. Ketone bisulfiteadduct, 76.0% w/w assay (NMR, using DMB as internal standard ind₆-DMSO), (1.00 eq, 1.00 wt corr. for w/w assay) for input calculation.

TABLE 1B Table for isolated yields for step 2 Demonstration batch % w/w% area Corr. Corr. Corr. Yield (¹H- (HPLC, Input Output (% th) NMR)* QC)Comments 1200.0 g 858.15 g 108.3 25.5 99.31 Purified ketone

Step 3: Preparation of Fenfluramine HCl Crude

Procedure: Charge the ZX008 ketone (corr. for assay, 1.00 wt, 1.00 eq.isolated as solution in MeOH in stage 2) to a vessel. Charge methanol,code RM0036 (5.0 vol, 4.0 wt) to the mixture at 18 to 23° C. Cool thesolution to 0 to 5° C. Charge 70 wt % aqueous ethylamine solution (1.3vol, 1.6 wt, 4.0 eq) to the mixture at 0 to 10° C., over 15 to 30minutes, followed by a line rinse with methanol (1.0 vol, 0.8 wt). Warmthe mixture to 15 to 20° C. and stir the mixture for a further 60 to 70minutes at 15 to 20° C. Adjust the mixture to 15 to 18° C. if required,targeting 15° C. Charge sodium triacetoxyborohydride (2.4 wt, 2.25 eq.)to the mixture in approximately 10 portions, keeping the mixture at 15to 20° C., targeting 17° C. Addition time 1.5 to 2 hours. Caution:Exothermic. Stir the mixture at 15 to 20° C. until complete by HPLCanalysis, pass criterion ≤3.0% area ZX008 ketone, typically 2 to 3hours. Adjust the pH of the mixture to pH>12 by charging 20% w/w aqueoussodium hydroxide solution (5.0 to 6.0 vol) to the mixture at 15 to 40°C. Addition time 10 to 30 minutes. Caution: Exothermic. If necessary,adjust the temperature to 18 to 23° C. Extract the mixture with toluene(3×3.0 vol, 3×2.6 wt) at 18 to 23° C., retaining and combining the toporganic layer after each extraction. Wash the combined organic layerwith purified water, (1.0 vol) at 18 to 23° C. Heat the mixture to 40 to50° C., targeting 48° C. Concentrate the mixture under reduced pressureat constant volume maintaining ca. 5 vol by charging the organic layerat approximately the same rate as the distillation rate at 40 to 50° C.,targeting 48° C. Cool the mixture to 18 to 23° C. Charge purified water(10.0 vol) to the mixture at 18 to 23° C. Adjust the pH of the mixtureto 0.1≤pH≤1.5 at 18 to 23° C. by charging concentrated hydrochloricacid, 0.5 vol. Do not delay from this step until neutralization.

Separate the layers at 18 to 23° C. retaining the bottom aqueous layer.Wash the aqueous layer with toluene, (3.0 vol, 2.6 wt) at 18 to 23° C.retaining the aqueous layer. Adjust the pH of the aqueous layer to pH>12by charging 20% w/w sodium hydroxide solution at 18 to 23° C. 0.8 to 0.9vol. Caution: Exothermic. Charge TBME, code RM0002 (2.0 vol, 1.5 wt) tothe basic aqueous layer. Separate the layers at 18 to 23° C. retainingthe top organic layer. Back-extract the aqueous layer with TBME (2×2.0vol, 2×1.5 wt) at 18 to 23° C. retaining the organic layers. Wash thecombined organic layer with purified water, (2×1.0 vol) at 18 to 23° C.Concentrate the combined organic layers under reduced pressure at 40 to50° C., targeting 48° C. to ca. 3 vol. Determine the residual toluenecontent of the mixture by 1H-NMR analysis. Sample for determination ofresidual water content by KF analysis, AKX reagent. Charge TBME (8.7vol, 6.4 wt) to the mixture at 40 to 50° C. Cool the solution to 0 to 5°C., targeting 2° C. Charge concentrated hydrochloric acid (0.54 vol,0.46 wt) maintaining the temperature <15° C. Caution: Exothermic. Linerinse with TBME (1.0 vol, 0.7 wt). If necessary, adjust the temperatureto 0 to 10° C. and stir the mixture at 0 to 10° C. for a further 2 to 3hours. Filter the mixture and wash the filter-cake with TBME (2×4.4 vol,2×3.3 wt) at 0 to 10° C. Dry the solid at up to 40° C. until the TBMEcontent is ≤0.5% w/w TBME by 1H-NMR analysis. 4 to 8 hours.

Yields and Profiles: The yield for the step 3 Demonstration batch issummarized in Table 1C below. Input: 856.8 g corr. Ketone, 44.2% w/wassay (NMR, using TCNB as internal standard in CDCl₃), (1.00 eq, 1.00 wtcorr. for w/w assay) for input calculation. FIG. 2 and Table 1D shows anexemplary HPLC chromatogram of a crude preparation of fenfluraminehydrochloride (210 nm UV absorbance).

TABLE 1C Table for isolated yields for step 3 Demonstration batch % w/w% area Reference Corr. Corr. Corr. Yield (¹H- (HPLC, number Input Output(% th) NMR)* QC) Comments Batch A1 856.8 g 836.31 g 85.3 44.2 99.15Fenfluramine free base (in situ intermediate) Batch A2 880.7 84.0 basedon 99.5 100.00 Fenfluramine•HCl ketone crude (step 3 and bisulfite 4.1)adduct (77.6 based on purified ketone)

TABLE 1D Purity of crude fenfluramine hydrochloride by HPLC (see FIG. 2)Processed Channel Descr. DAD AU Ch 1 Sample 210, Bw 4 Peak Results USPUSP USP Name RT RelRT Area Height Tailing Resolution Plate Count EP s/n% Area 1 NorFenfluramine 7.46 2 2-Fenfluramine 7.68 3 Fenfluramine 8.671.000 3789064 778178 1.7 70796 2549.8 99.15 4 4-Fenfluramine 8.95 511.34 1.308 6073 1449 1.2 23.5 215529 3.8 0.16 6 ZX008 acid 12.93 7Fenfluramine alcohol 14.16 1.633 15266 2972 1.3 24.8 215040 8.7 0.40 8ZX008 ketone 14.83 9 Fenfluramine acetamide 15.55 10 TOLUENE 15.75 1115.92 1.836 4110 1122 2.7 0.11 12 16.60 1.915 6861 1630 1.5 451209 4.30.18 Sum 3821374 100.00

Step 4.2: Crystallization of Fenfluramine Hydrochloride

Procedure: Charge Fenfluramine.HCl (crude) (1.00 wt, 1.0 eq.) and TBME(10.0 vol, 7.4 wt) to the vessel and commence stirring. Heat thesuspension to reflux (50 to 58° C.). Charge ethanol (5.0 vol, 3.9 wt)maintaining the temperature at 50 to 58° C. Addition time 20 minutes.Stir at 50 to 58° C. for 5 to 10 minutes and check for dissolution. Stirthe solution at 50 to 58° C. for 5 to 10 minutes, targeting 54 to 58° C.Clarify the reaction mixture through a 0.1 μm in-line filter at 54 to58° C., followed by a line rinse with TBME (1 vol, 0.7 wt). Cool thesolution to 48 to 50° C. Charge Fenfluramine HCl, code FP0188 (0.01 wt).Check for crystallization. Allow the suspension to cool to 15 to 20° C.,target 17° C. over 5 to 5.5 hours at an approximately constant rate.Stir the mixture at 15 to 20° C., target 17° C. for 2 to 3 hours. Filterthe mixture and wash the filter-cake with clarified TBME (2×3.0 vol,2×2.2 wt) at 5 to 15° C. Dry the solid at up to 40° C. until the TBMEcontent is ≤0.5% w/w TBME and the ethanol content is <0.5% w/w EtOH by1H-NMR analysis. 4 to 8 hours. Determine the w/w assay of the isolatedFenfluramine.HCl by 1H-NMR analysis.

Yields and Profiles: The yield for the stage 4 Demonstration batch issummarized in Table 1E below. Input: 750.0 g uncorr. Fenfluramine HClcrude (1.00 eq, 1.00 wt uncorr.) for input calculation. FIG. 3 shows anexemplary HPLC chromatogram of a crystallized fenfluramine hydrochloridesample (210 nm UV absorbance).

TABLE 1E Table for isolated yields for stage 4 Demonstration batchUncorr. Uncorr. Uncorr. Yield HPLC (% area, Input Output (% th) QC)Comments 750.0 g 608.0 81.1 100.00* Fenfluramine•HCl5. In-Process Controls

Table 2 summarizes the in-process controls (IPCs) by IPC number as citedin the narrative procedures above used for Process 1.

TABLE 2 In-Process Controls Performed during Process 1 Critical IPCSynthesis Process No. Step Sample Description Method Acceptance Criteria1 1 Reaction Reaction HPLC NMT 3.0% Ketone (1) Mixture Completion 2 1Extraction Purity HPLC Report percent Aqueous Fenfluramine Free Base andPhase Fenfluramine Alcohol 3 1 Extraction Purity HPLC Report percentOrganic Fenfluramine Free Base and Phase Fenfluramine Alcohol 4 1Extraction Purity HPLC Report percent Organic Fenfluramine Free Base andPhase Fenfluramine Alcohol 5 1 Extraction Purity HPLC NLT 98.0%Fenfluramine Aqueous HCl Phase LT 1.0% Fenfluramine Alcohol 6 1Extraction Purity HPLC Report percent result of Aqueous Fenfluramine HClPhase Fenfluramine Alcohol 7 2 Reaction Purity ¹H-NMR Residual Solventsby ¹H- Mixture NMR Ethanol NMT 0.50% w/w Ethyl Acetate NMT 0.50% w/wMethanol NMT 0.50% w/w Toluene NMT 0.50% w/w MTBE NMT 0.50% w/w6. Starting Materials

This section provides information and specification controls for thestarting materials used to produce clinical supplies of fenfluramine perthe routes shown herein.

TABLE 3 Starting Materials via the Route 1 Chemical Name [CAS. No.] CodeName Structure Source Step 1-(3- (Trifluoromethyl)- phenylacetone[21906-39-8] Ketone (1)

Fluorochem 1 Ethyl Amine (70% in water) [75-04-7] Ethyl Amine EtNH2 AlfaAesar 1

TABLE 4 Starting Materials via Route 2 Chemical Name [CAS. No.] CodeName Structure Source Step 3-(Trifluoromethyl)- phenylacetic acid[351-35-9] Acid (1a)

To be determined 1 Acetic Anhydride [108-24-7] Acetic Anhydride

Various 1 Ethyl Amine (70% in water) [75-04-7] Ethyl Amine EtNH2 Various3

Table 5 provides a list of the intermediates for the Route 2 synthesis.Both routes share the same intermediate Fenfluramine Free Base (1).Fenfluramine Free Base (1) was treated as an isolated intermediate inthe Route 1 process however the Route 2 process uses fixed equipmentwhere both Ketone (2) and Fenfluramine Free Base 1, both non-isolatableoils, are telescoped as a solution and controlled as in-situintermediates. The Bisulfate Complex (3) is isolated as a solid thus isamenable to treatment as an isolated intermediate and released as such.Crude Fenfluramine HCl can be isolated as an intermediate beforerecrystallization.

A Specification and Testing Strategy for Intermediates is used.Additional tests and acceptance criteria are be added based upon reviewof data from the primary stability batches and process validationcritical parameter studies. Analytical reference standards are used infull characterization of each intermediate. HPLC methods to determineassay and impurities are the same as the drug substance release methodand are validated for Accuracy, Precision: Repeatability, IntermediatePrecision, Selectivity/Specificity, Detection limit, Quantitation limit,Linearity, Range, and Robustness.

TABLE 5 In-Situ and Isolated Intermediates Chemical Name [CAS No] CodeName Step No. Control Structure Bisulfate Complex of Ketone 1 BisulfateComplex (3) Step 1 Isolated (Solid)

1-(3- (Trifluoromethyl)- phenylacetone [21906-39-8] Ketone (2) Step 2In-Situ (oil)

Fenfluramine Free Base [458-24-2] Fenfluramine Free Base (1) Step 3In-Situ (oil)

Fenfluramine HCl [404-82-0] Crude Fenfluramine HCl Step 4 Isolated(Solid)

7. Characterization

Physiochemical Characteristics of Drug Substance.

Fenfluramine HCl is developed as a single polymorph Form 1. Apolymorphism and pre-formulation study has been conducted. Under a widerange of solvents and conditions crystalline material is produced of thesame polymorph Form 1 based on a well-defined XRPD pattern and aconsistent reproducible endotherm by DSC analysis. A summary of thechemophysical properties of Fenfluramine HCl from this study is providedbelow. Tabulated data includes example diffractograms, DSCs, andmicrographs.

The input Fenfluramine HCl (from precipitative isolation) wascharacterized to provide reference data and also to determine if thesalt was of the same form as that identified from previous saltformations. The XRPD pattern of the salt reveals a crystalline solidthat visually matches the reflection patterns obtained from formalcrystallization of Fenfluramine HCl and has been arbitrarily termedForm 1. Comparison of the μATR-FTIR data for the salt from variousbatches gave profiles that had a 99.95% match.

Thermal data analysis matched previous data obtained with only one majorendotherm on the DSC thermograph peaking at 172.3° C. that matches thebeginning of potential decomposition shown in a TGA thermograph. Thisalso matches the reported melting point quoted for the referencestandard.

Isolation of the amorphous form has been shown to be difficult, withattempts using three common methods (rapid solvent evaporation,anti-solvent precipitation and lyophilization) all yielding highlycrystalline solids that very closely share the same XRPD pattern of theinput Form 1.

Stability analysis of the salt after one week at 40° C./0% RH, threeweeks at 40° C./75% RH, and under photostability conditions revealedthat the input Form 1 has been maintained with no new impuritiesobserved at 0.1% threshold.

Results from DSC heat cycling analysis of Fenfluramine HCl arecomparable to results generated when the material was held at 170° C. Nocrystallization event was noted and the amorphous was not generated butrather Form 1 was returned.

Holding Fenfluramine HCl at approximately 170° C. for several hourscauses a melt and evaporation event to take place with recombination andcooling to provide a white solid. Analysis of the white solid by XRPD,DSC and ¹H NMR indicates no change in chemical or physical form, purity,or dissociation.

Forced degradation studies carried out have proven Fenfluramine HCl tobe stable under a range of conditions. Thermal modulation ofFenfluramine HCl repeatedly yielded the input Form 1.

8. Impurities

Impurities in a drug substance can be organic impurities (processimpurities or drug substance-related degradants), inorganic impurities(salt residues or metals) and residual solvents; some of theseimpurities must be evaluated as to whether or not they are genotoxicagents. These impurities are taken into consideration and controlled inFenfluramine HCl preparation by using either compendia or validatedanalytical methods per the specifications or by separate “forinformation only” testing. The following sections address the actual andpotential impurities in Fenfluramine HCl.

Actual Impurities and the Qualification of Synthesis Batch

No impurities reported in cGMP drug substance batches intended for usein humans have exceeded the ICHQ3A qualification thresholds of 0.15%(Table 8). All impurities >0.1% are identified and handled as describedin ICH Q3A unless they are genotoxic impurities.

Process Impurities

Table 6 lists the known potential impurities arising from the route ofsynthesis. All of these impurities are controlled to below ICHQ3Aqualification threshold of 0.15% by either process changes and/orcontrol of starting material input purities.

TABLE 6 Fenfluramine HCl Known Potential Process Impurities (Route 1)Observed Observed in in Development cGMP Name PLC Batches Batches [Cas.No.] Source (RRT) ≥0.10%¹⁾ ≥0.10%¹⁾ Ketone (2) Starting RRT No No[351-35-9] Material or 0.89 Intermediate Fenfluramine By-product RRT YesNo Alcohol 1.60 [621-45-4] Norfenfluramine By-product RRT Yes Yes[1886-26-6] 1.67 2-Fenfluramine Starting RRT No No [172953-70-7]Material 0.89 (isomer) 4-Fenfluramine Starting RRT Yes Yes [1683-15-4]Material 1.02 (isomer) N-(3- By-product RRT Yes Yes (trifluoromethyl)-0.53-0.57 benzyl)ethanamine [90754-95-3] ¹⁾ICH Q3A Identificationthreshold. The Reporting threshold (LOQ) for the HPLC method is 0.05%.

Degradation Impurities

No change in impurity profile is observed upon long-term storage basedon forced degradation studies under the ICH Q1A(R2) conditions of heat(solid, solution), acid, base, oxidizing, and ICH Q1B photostabilityconditions (solid, solution). Fenfluramine HCL is stable for 7 days as asolid at 150° C. (99.90 parent area %), as a solution inwater-acetonitrile at 70° C. (99.73 parent area %), as a solution inacid, base, or photosensitizing conditions at ambient. Only oxidizingconditions (peroxide conditions) produced degradation of FenfluramineHCl to 94.42% after 1 day producing several new related substances at˜1% each consistent by LC-MS with +16 oxidation by-products

Organic Volatiles/Residual Solvents

Table 11 in the Batch Analysis section summarizes the solvents used inthe process and the resulting amounts found in drug substance. Allsolvents used in the GMP steps are controlled at ICH Q3A limits using asuitably qualified Head-Space (HS) GC method.

Inorganic Impurities

Heavy Metals conform to either USP <231> or ICP method USP <233> as wellas ICH Q3D.

Genotoxic Impurities

The ICH guidelines Q3A and Q3B are not sufficient to provide guidance onimpurities that are DNA-reactive. The European Medicines Agency (EMA)guideline (2006) “Guideline on the Limits of Genotoxic Impurities” (EMA2006) and the ICH Guideline M7 (2014) “Assessment and Control of DNAReactive (Mutagenic) Impurities in Pharmaceuticals to Limit PotentialCarcinogenic Risk” (ICH Guideline M7) are taken into consideration incontrolling for potential genotoxic impurities. The diazonium route toprepare ketone (2) described in FIG. 5 has a disadvantage due to thepotential formation of genotoxic intermediates shown as boxed compounds(e.g., N-hydroxyaryl, N-nitrosamine and Nitro compound). Muller et al.(Regulatory Toxicology and Pharmacology 44 (2006) 198-211) listpotential functional alert groups that can be genotoxic. Safetyguidances and regulations indicate that analysis of a process andidentification of potential genotoxic agents, and control of suchimpurities at sub 10 parts per million levels is critical for safety.Often removal of such impurities and/or demonstrating their absence iscostly and time consuming and sometimes difficult to achievetechnically. For these reasons, selecting synthetic routes thatcircumvent the potential for such toxic intermediates is important.Because of the potential problems with the diazo route discussed above,as well as potential safety issues using diazo (shock-sensitive)intermediates, as well as the lower purity profiles with this route,this route is less preferred than the preferred route to ketone (2)starting from Nitrile (5). This route produces no potential genotoxicagents and leads to high purity Ketone (2) after isolation bydistillation or via the bisulfite salt adduct—hydrolysis sequence.

Additionally, attempts to remove isomer by-products present incommercial supplies of Aniline were unsuccessful whereas crystallizationthe Acid (4) resulting from hydrolysis of the nitrile (5) providescrystalline Acid (4) which can be purified to remove isomers early insynthesis. Removing impurities and/or isomers early in a synthesis ispreferred if it is known such impurities track to final product, as theneed to crystalize a final product at the end of a synthesis is morecostly in losses and impacts cost of goods more greatly than removingearly in synthesis before raw materials are invested along the process.

TABLE 7 Potential Impurities in Fenfluramine Synthesis Synthesis RouteNo. Compound Route 1 Route 2 CAS. No. 1

No Starting Material [351-35-9] 2

Starting Material Intermediate [21906-39-8] 4

No Intermediate Not Available 5

Potential Impurity Potential Impurity [621-45-4] 6

Potential Impurity Potential Impurity [1886-26-6] 7

Potential Impurity Potential Impurity [172953-70-7] 8

Potential Impurity Potential Impurity [1683-15-4] 9

Potential Impurity Potential Impurity [90754-95-3]

TABLE 8 Batch Analyses of Fenfluramine HCl Drug Substance Test Batch 1Batch 2 Batch 3 Batch 4 Appearance* White solid White solid White solidWhite solid Identification: FTIR* ^(a)) ^(a) Conforms ConformsIdentification: ¹H-NMR Conforms Conforms Conforms ConformsIdentification: ¹³C-NMR Conforms Conforms Conforms ConformsIdentification: MS Conforms Conforms Conforms Conforms Purity (HPLC area%) 99.57 99.77 ^(b)) ^(b) Assay (w/w %)* 99.49 100.37 100.79 100.13Anhydrous Basis (HPLC) Impurities 2-Fenfluramine ND ND ND ND (HPLC4-Fenfluramine) 0.16 0.15 0.11 0.12 area %) Fenfluramine Alcohol ND NDND ND 1-((3-trifluoromethyl)phenyl)acetone ND ND ND ND Acetamide 0.27 NDND ND N-(3-(trifluoromethyl)- ND 0.08 0.07 0.13 benzyl)ethanamine (RRT0.53-0.57) Total 0.43 0.23 0.19 0.25 Residual Solvents Methanol ND ND NDND (GC): ppm Ethanol ND ND ND ND MTBE 597 844 472 800 Ethyl Acetate 115164 79 150 Toluene 4 7 ND ND Residue on Ignition (w/w %) 0.01 0.02 0.04ND Heavy Metals (as Pb) <10 ppm <10 ppm ^(b) ^(b) Heavy Metals ICP (ppm)As ^(a) ^(a) <0.1 <0.1 Cd ^(a) ^(a) 0.1 0.1 Hg ^(a) ^(a) <0.1 <0.1 Pb^(a) ^(a) 0.2 <0.4 Co ^(a) ^(a) <0.1 0.1 Mo ^(a) ^(a) <0.1 <0.1 Se ^(a)^(a) <0.1 <0.1 V ^(a) ^(a) <0.1 <0.1 Water Determination* 0.21 0.08 0.020.03 (Karl Fischer) Chloride content by titration 13.19 13.09 12.9212.93 XRPD* Form 1 Form 1 Form 1 Form 1 Differential Scanning Onset169.42° C. 169.23° C. 169.85° C. 168.70° C. Calorimetry (DSC)* Peak172.82° C. 171.55° C. 172.22° C. 171.97° C. Particle Size % Volume mean(D) ^(a) 11 11 19 Malvern (μm) D10 ^(a) 1 1 1 D50 ^(a) 5 7 9 D90 ^(a) 1726 32 Microbial Total aerobic ^(a) ^(a) LT 100 CFU/g LT 100 CFU/g LimitsTests* microbial Count USP <61> Total yeast and ^(a) ^(a) LT 100 CFU/gLT 100 CFU/g molds count USP <62> Absence of ^(a) ^(a) Absent AbsentPathogens ^(a)These tests were added to the specifications recently thusonly recent lots have been tested using this test. ^(b)These tests havebeen dropped from the specifications thus only historical lots have beentested using this test.

Example 3

Method for Hydrolysis of Nitrile (5) to Acid

TABLE 9 Step Operation 1. Charge 3-(trifluoromethyl)phenyl acetonitrile(1.0 eq., 1.00 wt) and purified water (5.0 vol) to a vessel and commencestirring. 2. Dissolve sodium hydroxide (1.1 wt, 5.0 eq.) in purifiedwater (4.0 vol) at up to 40° C. in a suitable make-up vessel. Cautionvery exothermic. 3. Charge the aqueous sodium hydroxide solution to themixture from step 1 at up to 40° C. followed by a line rinse withpurified water, code RM0120 (1.0 vol) at up to 40° C. 4. Heat themixture to 75 to 85° C., target 80° C. over 1 to 2 hours. 5. Heat themixture at 80° C. until ≤0.1% area nitrile by HPLC analysis, expected 4to 6 hours. 6. Cool the mixture to 18 to 23° C. 7. Adjust the pH of themixture to pH ≤2 by charging 6M HCl (expected 7.0 vol) to the mixture at18 to 23° C. Caution exothermic. 8. Stir the mixture for 15 to 30minutes at 18 to 23° C. 9. Filter and wash the filter-cake with purifiedwater (2 × 5.0 vol) at 18 to 23° C. 10. Slurry wash the filter-cake withn-heptane, code RM (2 × vol) at 0 to 5° C. 11. Dry the isolated solid atup to 45° C. until the water content is ≤.0.2% w/w by KF analysisaccording to MET/AN/0163, AKX-reagent. 12. Crystallization of crudestage 1 acid (1.00 wt for input calculation) 13. Charge the crude stage1 acid (1.00 wt), ethyl acetate (0.75 vol) and n-heptane (10.5 vol) to avessel and commence stirring. 14. Heat the mixture to 50° C. to achievedissolution. 15. Cool the mixture to 5° C. and age at 5° C. for at least30 mins. 16. Filter and wash the filter-cake with n-heptane (2 × 5.0vol). 17. Dry the isolated solid at up to 45° C. until the residualsolvent content by ¹H-NMR analysis is ≤.0% w/w EtOAc and ≤.0% w/wn-heptane. Expected yield: 60 to 90% th uncorr. 68 to 103% w/w Expectedpurity: 93.00 to 99% area by HPLC

Example 4

Evaluation of Minor Components Formed During Dakin-West Reaction inPreparation of Ketone (2)

The impurities formed during the Dakin-West chemistry and theirsubsequent removal using the distillation or via isolation of theproduct ketone as the bisulfite salt are described. The two majorimpurities found are shown below.

Table 10 shows a table of analytical data for crude Ketone (2) isolatedfrom Dakin-West reaction before and after bisulfite purification. Inentry 1 crude Ketone isolated directly from the Dakin-West step(pre-bisulfite treatment) is 61.66% purity (e.g. about 62%) and contains1.98% (e.g., about 2%) and 4.64% (e.g., about 5%) respectively ofimpurities having RRTs 1.20 and 1.34, which are proposed to be theacetate and dimer impurities (e.g., depicted above), respectively. Inentry 2 which is post bisulfite treatment these are other impurities areremoved leading to an overall purity of 95.55% (e.g., about 96%). Otherentries shown in Table 10 provide other examples of this impurityenhancement by bisulfite treatment of crude Dakin-West ketone. The lasttwo entries use pure Fluorchem ketone as input to the salt formationstep and re-isolation of ketone thus illustrating that the saltformation and re-isolation does not produce any impurities itself.Additionally use of bicarbonate extraction procedure during reactionworkup provides an improvement in purity of the resulting composition asit serves to remove any unreacted acid. Crude Ketone (2) made by theDiazo route showed similar improvements in purity when treated withbisulfite and isolated.

TABLE 10 Analytical purity data for crude Ketone (2) isolated fromDakin-West reaction before and after bisulfite purification. RRT isrelative retention time (min) in chromatogram. RRT 0.93 1.00 1.009 1.06Entry 0.85 Aniline 0.95 0.99 Ketone 1.004 Nitrile 1.02 Acid 1.1 1.151.34 1.38 1 1.38 1.76 0.04 0.49 61.66 nd 0.29 0.29 0.26 1.98 0.66 4.640.14 2 0.82 nd nd nd 95.53 0.31 0.14 nd 0.23 0.01 0.10 0.43 0.27 3 nd ndnd nd 77.82 nd nd nd nd 3.12 0.01 7.76 6.16 4 nd nd nd nd 98.82 nd 0.63nd nd nd 0.02 0.30 0.22 5 0.08 nd nd 0.05 72.02 nd 0.02 nd nd 7.11 0.043.58 10.33 6 nd nd nd nd 99.49 nd 0.02 nd nd nd 0.02 0.11 0.24 7 0.150.23 nd nd 98.35 nd nd nd nd nd nd nd 0.24 8 nd nd nd nd 99.84 nd nd ndnd nd nd nd nd Entry 1 (Crude ketone from Route 1); Entry 2 (KetoneRoute 1 post bisulfite release); Entry 3 (Crude ketone using crudeacid); Entry 4 (Ketone using crude acid Post bisulfite); Entry 5 (Crudeketone using cryst. acid); Entry 6 (Crude ketone using cryst. acid postbisulfite); Entry 7 (Crude ketone using cryst. acid); Entry 8(Fluorochem ketone); Entry 9 (Fluorochem ketone post bisulfite).

Example 5

Additional Method for Preparation of1-(3-Trifluoromethyl)phenyl-propan-2-one

35 mL of water and 45 g of 37% (w/w) aqueous hydrochloric acid are putin a flask equipped with stirrer and dropping funnel. 24.25 Grams (0.151moles) of m-trifluoromethylaniline are added after having cooled to 10degree C. with an ice bath and then, at 5 degree C., an aqueous solutioncontaining 12.43 g (0.180 moles) of sodium nitrite in 150 ml of water isslowly added. The reaction mixture is stirred for 30 minutes and then ispoured during 30 minutes into a mixture made by 90 ml of water, 1.35 g(0.014 moles) of cuprous chloride, 2.30 g (0.013 moles) of cupricchloride dihydrate, 50 ml of acetone, 40.8 g (0.300 moles) of sodiumacetate trihydrate and 23 g (0.230 moles) of isopropenyl acetate whilekeeping the reaction temperature at 30 degree C. After further 30minutes of stirring, the reaction mixture is brought to 20 degree C., 50ml of methylene chloride are added and the two layers are separated.

The aqueous layer is discarded while the organic layer is concentratedunder vacuum until an oil is obtained which is treated with 35 g ofsodium metabisulfite, 70 ml of water and 150 ml of heptane understirring at room temperature for 12 hours. The suspension is filtered,the bisulfite complex is washed on the filter with 50 ml of heptane andthen suspended in a two-phase mixture made by 100 ml of methylenechloride and 150 ml of a 10% (w/v) aqueous solution of sodium hydroxide.The layers are separated after one hour of stirring at room temperature,the aqueous phase is discarded while the organic layer is washed withwater and evaporated under vacuum to give pure ketone.

Notwithstanding the appended claims, the disclosure set forth herein isalso defined by the following clauses:

Clause 1. A method of preparing a fenfluramine active pharmaceuticalingredient, the method comprising:

(a) hydrolyzing a 2-(3-(trifluoromethyl)phenyl)acetonitrile compositionto produce a 2-(3-(trifluoromethyl)phenyl)acetic acid composition;

(b) reacting the 2-(3-(trifluoromethyl)phenyl)acetic acid compositionwith acetic anhydride and a catalyst to produce a1-(3-(trifluoromethyl)phenyl)propan-2-one composition; and

(c) reductively aminating the 1-(3-(trifluoromethyl)phenyl)propan-2-onecomposition with ethylamine using a borohydride reducing agent toproduce a fenfluramine composition.

Clause 2. The method of clause 1, wherein the2-(3-(trifluoromethyl)phenyl) acetonitrile composition comprises atleast 0.2% by weight of trifluoromethyl-phenyl regioisomers.

Clause 3. The method of any one of clauses 1 and 2, wherein the2-(3-(trifluoromethyl)phenyl)acetonitrile composition is prepared fromtrifluoromethylbenzene.

Clause 4. The method of any one of clauses 1-3, further comprising,prior to step (b), purifying the 2-(3-(trifluoromethyl)phenyl)aceticacid composition to produce a composition substantially devoid of one ormore trifluoromethyl-phenyl regioisomers and substantially devoid oftrifluoromethylbenzaldehyde and benzaldehyde.

Clause 5. The method of clause 4, wherein the purifying comprisescrystallization of 2-(3-(trifluoromethyl)phenyl)acetic acid from thecomposition.

Clause 6. The method of any one of clauses 1-5, wherein step (b)comprises purification of the 1-(3-(trifluoromethyl)phenyl)propan-2-onecomposition via a ketone bisulfite adduct.

Clause 7. The method of any one of clauses 1-6, wherein step (b)comprises selectively reacting 2-(3-(trifluoromethyl)phenyl)acetic acidin the presence of unreacted 2-(2-(trifluoromethyl)phenyl)acetic acid.

Clause 8. The method of any one of clauses 1-7, wherein step (b) furthercomprises removing unreacted 2-(2-(trifluoromethyl)phenyl)acetic acidregioisomer from the 1-(3-(trifluoromethyl)phenyl)propan-2-onecomposition.

Clause 9. The method of any one of clauses 1-8, wherein the fenfluraminecomposition is crude and substantially devoid of: trifluoromethyl-phenylregioisomers of fenfluramine or a salt thereof; metal catalysts; Class Iand/or Class II solvents (ICH Q3C) (e.g., Benzene, Carbon tetrachloride,1,2-Dichloroethane, 1,1-Dichloroethene and/or 1,1,1-Trichloroethane);and a reduced alcohol side product.

Clause 10. The method of any one of clauses 1-9, wherein thefenfluramine composition is crude and has less than 1% by weight intotal of trifluoromethyl-phenyl regioisomers of fenfluramine or a saltthereof.

Clause 11. The method of any one of clauses 1-10, wherein theborohydride reducing agent is sodium triacetoxyborohydride.

Clause 12. The method of any one of clauses 1-11, wherein thefenfluramine composition is crude and has less than 10% by weight of areduced alcohol side product.

Clause 13. The method of any one of clauses 1-12, further comprisingcrystallizing fenfluramine or a salt thereof from the fenfluraminecomposition.

Clause 14. The method of clause 1, wherein step (a) is performed underaqueous acidic conditions.

Clause 15. The method of clause 14, wherein the yield of step (a) is 80%or more.

Clause 16. The method of clause 1, wherein step (b) is performed underconditions that include contacting the2-(3-(trifluoromethyl)phenyl)acetic acid composition with about 0.5equivalents of 1-methylimidazole and about 5 equivalents or more ofacetic anhydride in an optional solvent.

Clause 17. The method of clause 16, wherein the yield of step (b) is 80%or more.

Clause 18. The method of clause 1, wherein step (c) is performed underconditions that comprise contacting the1-(3-(trifluoromethyl)phenyl)propan-2-one composition with a solution of70% by weight of ethylamine in water and about 2.25 equivalents or moreof triacetoxyborohydride in methanol solvent.

Clause 19. The method of clause 18, wherein the yield of step (c) is 80%or more.

Clause 20. The method of clause 1, wherein the fenfluramine compositionhas following profile: at least 80% by weight of fenfluramine or a saltthereof; less than 1% by weight of 2-fenfluramine or a salt thereof;less than 1% by weight of 4-fenfluramine or a salt thereof; and lessthan 10% by weight of fenfluramine reduced alcohol side product.

Clause 21. The method of any one of clauses 1-20, further comprisingconverting fenfluramine in the fenfluramine composition to apharmaceutically acceptable salt of fenfluramine.

Clause 22. The method of clause 21, further comprising crystallizing thepharmaceutically acceptable salt of fenfluramine from the fenfluraminecomposition.

Clause 23. The method of clause 22, wherein the pharmaceuticallyacceptable salt of fenfluramine has following purity profile: at least90% or more of the pharmaceutically acceptable salt of fenfluramine;less than 1% by weight of 2-fenfluramine; less than 5% by weight of4-fenfluramine; and less than 5% by weight of fenfluramine reducedalcohol side product.

Clause 24. The method of any one of clauses 20-22, wherein thepharmaceutically acceptable salt of fenfluramine is fenfluraminehydrochloride.

Clause 25. The method of any one of clauses 1-20, further comprisingpurifying fenfluramine free base from the fenfluramine composition.

Clause 26. The method of any one of clauses 1-25, further comprisingperforming a chiral separation of a racemic fenfluramine composition toproduce a non-racemic fenfluramine composition comprising a predominantstereoisomer of fenfluramine.

Clause 27. The method of clause 26, wherein the predominant stereoisomerof fenfluramine is(S)—N-Ethyl-1-[3-(trifluoromethyl)phenyl]-propan-2-amine.

Clause 28. The method of clause 26, wherein the predominant stereoisomerof fenfluramine is(R)—N-Ethyl-1-[3-(trifluoromethyl)phenyl]-propan-2-amine.

Clause 29. A fenfluramine composition produced according to the methodof any one of clauses 1-28.

Clause 30. A fenfluramine active pharmaceutical ingredient comprising apharmaceutically acceptable salt of fenfluramine and having less than0.2% by weight in total of trifluoromethyl regioisomers.

Clause 31. The fenfluramine active pharmaceutical ingredient of clause30, having the following profile: at least 90% or by weight of apharmaceutically acceptable salt of fenfluramine; less than 0.2% byweight of 2-fenfluramine; less than 0.2% by weight of 4-fenfluramine;and less than 1% by weight of fenfluramine alcohol.

Clause 32. The fenfluramine active pharmaceutical ingredient of any oneof clauses 30-31, wherein heavy metal components are substantially orcompletely eliminated from the composition.

Clause 33. The fenfluramine active pharmaceutical ingredient of any oneof clauses 30-31, wherein Class 1 and/or Class 2 solvents aresubstantially or completely eliminated from the composition (e.g., thefenfluramine active pharmaceutical ingredient of any one of clauses30-31, is substantially devoid of Class 1 and/or Class 2 solvents).

Clause 34. The fenfluramine active pharmaceutical ingredient of any oneof clauses 30-33, wherein fenfluramine alcohol is completely eliminatedfrom the composition.

Clause 35. The fenfluramine active pharmaceutical ingredient of any oneof clauses 30-34, wherein benzaldehyde and trifluorobenzaldehyde aresubstantially or completely eliminated from the composition.

Clause 36. The fenfluramine active pharmaceutical ingredient of any oneof clauses 30-35, wherein the composition is unpurified.

Clause 37. A pharmaceutical composition, comprising the fenfluramineactive pharmaceutical ingredient of any one of clauses 30-36 and apharmaceutically acceptable excipient.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

That which is claimed is:
 1. A method of preparing a fenfluramine activepharmaceutical ingredient, the method comprising: (a) hydrolyzing a2-(3-(trifluoromethyl)phenyl)acetonitrile composition to produce a2-(3-(trifluoromethyl)phenyl)acetic acid composition; (b) purifying the2-(3-(trifluoromethyl)phenyl)acetic acid composition via crystallizationto produce a purified 2-(3-(trifluoromethyl)phenyl)acetic acid havingless than 0.2% by weight in total of trifluoromethyl-phenylregioisomers; (c) reacting the purified2-(3-(trifluoromethyl)phenyl)acetic acid composition with aceticanhydride and a catalyst to produce a1-(3-(trifluoromethyl)phenyl)propan-2-one composition; and (d)reductively aminating the 1-(3-(trifluoromethyl)phenyl)propan-2-onecomposition with ethylamine using a borohydride reducing agent toproduce a crude fenfluramine composition having less than 0.2% by weightin total of trifluoromethyl-phenyl regioisomers of fenfluramine or asalt thereof.
 2. The method of claim 1, wherein the2-(3-(trifluoromethyl)phenyl)acetonitrile composition comprises at least0.5% by weight of 4-trifluoromethyl-phenyl regioisomer.
 3. The method ofclaim 2, wherein step (c) comprises purification of the1-(3-(trifluoromethyl)phenyl)propan-2-one composition via a ketonebisulfite adduct.
 4. The method of claim 2, wherein the purified2-(3-(trifluoromethyl)phenyl)acetic acid composition of step (b) hasless than 0.1% by weight 4-trifluoromethyl-phenyl regioisomer.
 5. Themethod of claim 4, wherein the purified2-(3-(trifluoromethyl)phenyl)acetic acid composition of step (b) hasless than 0.1% by weight 2-trifluoromethyl-phenyl regioisomer.
 6. Themethod of claim 1, wherein the 2-(3-(trifluoromethyl)phenyl)acetonitrilecomposition is prepared from trifluoromethylbenzene.
 7. The method ofclaim 1, wherein the crude fenfluramine composition: has less than 0.2%by weight of trifluoromethyl-phenyl regioisomers of fenfluramine or asalt thereof; is devoid of metal catalysts; is devoid of solventsselected from acetonitrile, benzene and substituted benzenes, carbontetrachloride, chloroform, cyclohexane, 1,2-dichloroethane,1,1-dichloroethane, 1,2-dimethoxyethane, DMF, 1,4-dioxane, methanol,methylbutyl ketone, N-methylpyrrolidinone, pyridine, toluene,1,1,1-trichloroethane, 1,1,2-trichloroethene, and xylene; and has lessthan 5% by weight of reduced alcohol side product.
 8. The method ofclaim 1, wherein the crude fenfluramine composition has less than 0.2%by weight of 4-fenfluramine or a salt thereof.
 9. The method of claim 8,wherein the crude fenfluramine composition has less than 0.1% by weightof 4-fenfluramine or a salt thereof.
 10. The method of claim 9, whereinthe crude fenfluramine composition has less than 0.1% by weight of2-fenfluramine or a salt thereof.
 11. The method of claim 1, wherein thecrude fenfluramine composition has less than 10% by weight of a reducedalcohol side product.
 12. The method of claim 1, further comprisingcrystallizing fenfluramine or a salt thereof from the crude fenfluraminecomposition.
 13. The method of claim 1, wherein step (c) is performedunder conditions that comprise contacting the2-(3-(trifluoromethyl)phenyl)acetic acid composition with about 0.5equivalents of 1-methylimidazole and about 5 equivalents or more ofacetic anhydride in an optional solvent.
 14. The method of claim 1,wherein step (d) is performed under conditions that comprise contactingthe 1-(3-(trifluoromethyl)phenyl)propan-2-one composition with asolution of 70% by weight of ethylamine in water and about 2.25equivalents or more of triacetoxyborohydride in methanol solvent. 15.The method of claim 1, wherein the fenfluramine composition hasfollowing profile: at least 80% by weight of fenfluramine or a saltthereof less than 0.1% by weight of 2-fenfluramine or a salt thereof;less than 0.2% by weight of 4-fenfluramine or a salt thereof; and lessthan 10% by weight of fenfluramine reduced alcohol side product.
 16. Themethod of claim 1, further comprising: converting fenfluramine in thecrude fenfluramine composition to a pharmaceutically acceptable salt offenfluramine; and crystallizing the pharmaceutically acceptable salt offenfluramine, wherein the pharmaceutically acceptable salt offenfluramine has following purity profile: at least 95% of thepharmaceutically acceptable salt of fenfluramine; less than 0.1% byweight of 2-fenfluramine; less than 0.2% by weight of 4-fenfluramine;and less than 0.1% by weight of fenfluramine reduced alcohol sideproduct.
 17. The method of claim 1, further comprising purifyingfenfluramine free base from the crude fenfluramine composition.
 18. Themethod of claim 1, further comprising performing a chiral separation ofa racemic fenfluramine composition to produce a non-racemic fenfluraminecomposition comprising a predominant stereoisomer of fenfluramine.